This study was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) under IG 2017-ID. 20172 project &#8211; P.I. Corsi Fabio. SM acknowledges Pediatric Clinical Research center &#8220;Romeo and Enrica Invernizzi&#8221; that supports her position. AB thanks AIRC (ID. 20172 project) and University of Milan for research fellowship. LS and MS postdoctoral and doctoral fellowships are supported by University of Milan.

<ol>
	<li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Velaei, K., Samadi, N., Barazvan, B., Soleimani Rad, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment-mediated+chemoresistance+in+breast+cancer.">Tumor microenvironment-mediated chemoresistance in breast cancer.</a> Breast. 30, 92-100 (2016).</li><li>Cheng, J. D., Weiner, L. 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The authors have nothing to disclose.

CAFs are emerging as key players in remodeling extracellular matrix, promoting metastasis progression, and limiting drug access to the tumor site34. However, due to their heterogeneity, their roles are still controversial&#8212;some CAFs are tumorigenic, whereas some other subtypes seem to have a tumor-suppressive role. In this context, their isolation can be of extreme interest to shed more light on their much-debated role in cancer progression, which will have important clinical implications12,31. Moreover, successful CAF extraction from human and preclinical mouse tumor models would also facilitate the development of new CAF-targeting drugs. This paper reports a method to efficiently isolate and culture primary CAFs from a syngeneic preclinical model of breast cancer. Based on experience, three experimental steps mostly influence the success of the protocol.
The first one is working fast to avoid aggregation of cell suspensions associated with the risk of clotting in the column and slowing cell efflux: in fact, when cells that passed through the column were clustered together, the probability of a sub-optimal isolation process increased, and the final cultures contained &#8220;contaminant&#8221; cell clones. The second one is pooling cells from different tumors after the depletion passage to guarantee enough cells to be passed through in the final enrichment step. The final critical issue is plating the extracted cells at high cell densities, most likely starting from a single well of the 24-well plate to boost cell growth and colony expansion for up to 4&#8211;5 passages. If the cells were seeded at low densities, they expanded on the free surface and quickly stopped replicating.
Several studies have already described CAF extraction methods of both human and mouse origin, to study their role in promoting cancer development and invasiveness. This has been done in many types of tumors, including breast cancer, melanoma, cholangiocarcinoma, and pancreatic adenocarcinoma35,36,37,38. However, due to the lack of specific CAF markers and to their heterogeneity, the results of these processes are still sub-optimal.
Here, the isolated cells were used to validate the CAF-targeting ability of HFn nanocages functionalized with anti-FAP targeting moieties. The use of primary cultures of CAFs was a significant advantage, allowing the validation of the nanostrategy ex vivo with a much simpler and cost-effective binding experiment than doing it directly in vivo at this preliminary phase of nanodrug optimization.
In this sense, ex vivo testing on CAFs can precede in vivo animal experiments by allowing the screening and selection of the most promising nanoparticles for the optimal targeting of CAF. The CAF model presented here may also be used for preliminary efficacy studies, when loading nanoparticles with active drugs. Animals will be only used later to evaluate biodistribution, pharmacokinetics, and efficacy studies.
Several CAF-targeting nanodrugs are being developed, such as ferritin nanocages for photodynamic therapy in breast cancer and peptide-based particles to deliver doxorubicin in prostate cancer models39,40. However, not many studies have focused on the isolation of CAFs as cell platforms for nanodrug optimization.&#160;
This protocol has some limitations. First, it is a time-consuming protocol, requiring the preparation and purchase of several reagents and materials. Second, due to the scarcity of CAFs in the 4T1 breast tumor, their yield is low. Nanoparticle experiments should be planned and performed as soon as the required cell number is achieved, as primary CAFs undergo senescence and cannot be maintained in culture for a long time. In conclusion, this method of CAFs isolation, culturing, and characterization can be a powerful tool to accelerate the development of new targeted nanomedicines in the fight against cancer.

During the past decades, it has become clear that killing tumor cells is usually not sufficient to eradicate malignancy, as the tumor microenvironment may prompt tumor relapse and induce therapeutic resistance1,2. A novel paradigm has then emerged: targeting tumor stroma to deprive the tumor of supporting factors and thus, boost the efficacy of chemotherapeutics3,4,5. In particular, cancer-associated fibroblasts (CAFs) are an interesting stromal target in many solid tumors6,7. CAFs are a very heterogeneous group of cells that interact with cancer cells and cells of the immune system through the secretion of growth factors, cytokines, and chemokines; build up and remodel the extracellular matrix; and enable metastasis formation8,9,10,11,12. Depending on the tumor type, CAFs show pro-tumorigenic functions, while other subtypes of CAFs seem to have tumor-suppressive functions13,14. To better clarify this dichotomy, a thorough characterization of CAFs from primary and metastatic tumors is important.
In this context, an emerging field of research has focused on the development of nanosized agents designed to target and/or destroy CAFs by delivering active molecules and drugs able to remodel the tumor microenvironment15,16,17,18. Several types of nanoparticles have been designed to achieve CAF ablation by cytotoxic drugs, to induce CAF-targeted photodynamic therapy, or to reprogram CAFs by reverting them to a quiescent state or inducing TNF-related apoptosis induced ligand expression, which induces apoptosis of neighboring cancer cells16,19. Moreover, the potential of many nanoparticles to actively target specific biological markers gives rise to the hope of selecting CAF subsets to target. Although its absolute specificity for CAF is still questioned, fibroblast activation protein (FAP) is one of the most promising targets of pro-tumorigenic stroma and is exploited to steer nanodrug delivery, thus paving the path for the development of CAF-targeting nanotherapeutics20,21,22.
This paper describes the isolation of primary CAFs from a syngeneic model of murine breast cancer and reports their use in the study of the targeting capability of nanoparticles engineered to recognize the CAF marker, FAP. Ferritin nanocages are used as candidate nanosystems to set up the method, as their specificity of delivery may be shaped by the surface exposure of targeting moieties23,24. Moreover, ferritins have been successfully proven to be excellent biocompatible shuttles for antitumor applications, triggering rapid accumulation of the payload in the tumor mass25,26,27. To date, preclinical studies of CAF-targeting nanosystems have involved in vitro testing on fibroblast cell lines stimulated in culture with transforming growth factor-beta to induce cell activation and the expression of some immunophenotypic features of CAFs28,29. This method is usually applied to immortalized cell lines (such as NIH3T3, LX-2) and is quite rapid and simple, yielding activated cells in a few hours or days. A limitation is that although in vitro stimulation induces the expression of some genes attributed to activated myofibroblasts, it cannot entirely recapitulate all the biological features of real CAFs, especially their heterogeneity in vivo.
Another strategy involves the extraction of primary CAFs from human or mouse tumor samples30,31. This ensures that CAF activation occurs in a physiological context, and that the heterogeneity of CAF subpopulations is maintained. According to the research objective, CAFs may be derived from different sources, thus offering the possibility to study the most reliable condition. The protocol reported here would be valuable for scientists who seek to perform a preliminary evaluation of the functionality of novel nanoparticles designed to target CAFs from a murine breast cancer model. Isolated CAFs would be useful for screening those nanoparticles that are promising enough to proceed for in vivo evaluation in animal models of cancer. This will be relevant during the first steps of nanoparticle production, driving nanotechnologists toward the refinement of nanoparticle design by mainly considering the strategy of ligand immobilization to achieve optimal targeting properties.&#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACK Lysing buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Store at +15&#176; to +30 &#176;C.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-human antibody</td>
			<td>Immunological Science</td>
			<td>IS-20022</td>
			<td>Protect from light.</td>
		</tr>
		<tr>
			<td>Anti-FAP Fab fragment (3F2)</td>
			<td>Creative Biolabs</td>
			<td>TAB-024WM-F(E)</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Charles River</td>
			<td>028BALB/C</td>
			<td>7 weeks old female mice</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>CD45 antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-110-796</td>
			<td>FITC-conjugated fluorescent antibody; clone REA 737; Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>CD90.2 antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-960</td>
			<td>PE-conjugated fluorescent antibody; clone 30-H12; Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>Cell strainers 70 &#181;m</td>
			<td>VWR</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>laser configuration B4-R2-V0</td>
		</tr>
		<tr>
			<td>Dead cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td>contains 10 mL of Dead Cell Removal MicroBeads; 25 mL of 20&#215; Binding Buffer Stock Solution. Store protected from light at 2&#8722;8 &#176;C.</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td>reagent for in vivo imaging</td>
		</tr>
		<tr>
			<td>DMEM High Glucose w/o L-Glutamine&#160; w/ Sodium Pyruvate</td>
			<td>Euroclone</td>
			<td>ECB7501L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS w/o Calcium, w/o Magnesium</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Keep at room temperature.</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td>Before use, heat at 56 &#176;C for 30 min to kill all the complement proteins.</td>
		</tr>
		<tr>
			<td>Fluorescein Istothiocyanate isomer I (FITC)</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>GentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td>They are used in combination with the gentleMACS Dissociator.</td>
		</tr>
		<tr>
			<td>GentleMACS dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Two samples can be processed in parallel. Special protocols have been developed for various tissues.&#160;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Euroclone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12&#160; w/o L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB7502L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>This imaging system is easy to use for both fluorescent and bioluminescent imaging in vivo. Equipped with a Living Image Software for analysis</td>
		</tr>
		<tr>
			<td>LD columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td>Composed of ferromagnetic spheres, designed for stringent depletion of unwanted cells. Position the required number of columns on the magnetic Separation Unit and equilibrate with buffer before use</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td>200 mM stock</td>
		</tr>
		<tr>
			<td>Light/fluorescence microscope equipped with camera</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED Fluo/ ICC50 W CameraModule</td>
			<td>inverted microscope for live cells with camera</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Unit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>Position the magnet on appropriate stand before use.</td>
		</tr>
		<tr>
			<td>MACS Tissue Storage Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-008</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>Composed of ferromagnetic spheres, designed for positive selection of cells. Position the required number of columns on the magnetic Separation Unit and equilibrate with buffer before use.</td>
		</tr>
		<tr>
			<td>Mycoplasma Removal Agent</td>
			<td>Euroclone</td>
			<td>ECMC210A</td>
			<td>Dilute in culture medium 1:100.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Invitrogen</td>
			<td>A10235</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Non essential aminoacids</td>
			<td>Euroclone</td>
			<td>ECB3054D</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human apoferritin H-homopolymer (HFn)&#160;</td>
			<td>Molirom</td>
			<td>MLR-P018</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>Tumor-Associated Fibroblast Isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-116-474</td>
			<td>Contains 1 mL of Non-Tumor-Associated Fibroblast Depletion Cocktail, mouse; 1 mL of CD90.2 (Tumor-Associated Fibroblast) MicroBeads,mouse. Store protected from light at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-730</td>
			<td>Contains lyophilized enzymes (D, R, A) and buffer A; reconstitute enzymes according to the manufacturer&#39;s instructions, aliquot and store at - 20 &#176;C.</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>dilute 1:1 with a sample of cell suspension before counting the cells</td>
		</tr>
		<tr>
			<td>TrypLE select</td>
			<td>Gibco</td>
			<td>12563-029</td>
			<td>use at room temperature</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Lonza</td>
			<td>BE17-161E</td>
			<td>Warm at 37 &#176;C in a water bath before use</td>
		</tr>
		<tr>
			<td>T75 Primo TC flask</td>
			<td>Euroclone</td>
			<td>ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns</td>
			<td>Thermo Fisher Scientific</td>
			<td>89890</td>
			<td>7K MWCO, 2 mL</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Biosigma</td>
			<td>CL15.002.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Euroclone</td>
			<td>ET5015B</td>
			<td></td>
		</tr>
		<tr>
			<td>4T1-luc2</td>
			<td>Caliper</td>
			<td></td>
			<td>Mouse mammary gland cancer cell line stably transfected with firefly luciferase gene</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Euroclone</td>
			<td>ET5050B</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary cancer-associated fibroblasts from a syngeneic murine model of breast cancer for the study of targeted nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

In vivo model set-up for optimal CAFs isolation&#160; 
The injection of 105 4T1-luc cells into the mammary fat pad of female BALB/c mice leads to the growth of a detectable tumor mass at 5 days after implantation. By measuring the tumor volume by calipers and the tumor cell viability by BLI, tumor growth was monitored for one month after implantation. To find a sacrifice window that is adequate for CAFs isolation, an optimal compromise was sought between higher tumor size and BLI on the one hand and an emerging tumor ulceration and necrosis on the other hand (Figure 1). As a necrotic core appears 20 days after implantation, and it enlarges at 25 and 30 days (as documented by BLI images in Figure 1C), day 20 was set as the time point to optimize cell recovery after the isolation process. Even after carefully removing all visible necrotic areas during the first steps of tumor handling ex vivo, a high percentage of dead cells was found at the end of dissociation into single cells (Table 1). As this percentage may be relevant, especially with the increase in tumor size, dead cell removal is always necessary when working with the 4T1 model.
Optimization of CAFs isolation procedure, culture, and characterization 
Two more passages are needed to isolate the population of CAFs (CD90.2+ CD45-) from the panel of collected viable cells: the depletion of non-tumor-associated fibroblasts and the enrichment of tumor-associated fibroblasts (Figure 2). The depletion bead cocktail efficiently removes CD45+ cells (accounting for 67.35% and 0.69% of total cells pre- and post-depletion, respectively, Table 2), and was always used to process one single tumor in every column. As shown in Table 1, the number of eluted cells dropped from an average of 3 &#215; 106 to 1 &#215; 105 after the depletion step. Due to this massive decrease in total cell number, it is convenient to pool the collected cells from at least 2 to a maximum of 4 tumors in a single tube before proceeding with the enrichment step. By doing so, an adequate number of cells was obtained for incubation with tumor-associated fibroblast beads and passed through a single separation column to obtain a final average of 93% of CD90.2+ CD45- cells (Table 2).
Once seeded onto tissue culture plates, these recovered cells attached to the plastic and revealed a large spindle-shaped morphology typical of fibroblasts (Figure 3A,B) and different from 4T1 tumor cells (Figure 3D). Not pooling tumors after depletion causes the cellular yield with tumor-associated fibroblast microbeads to be too low to establish a culture. In other cases, when the duration and temperature of the incubations with microbeads are not carefully maintained, some non-specific binding may occur. In such cases, the enrichment steps were less efficient and higher percentages of CD90.2- CD45+ and CD90.2- CD45- cells were recovered along with the CD90.2+ CAFs (5.97 &#177; 1.5 and 16.75 &#177; 1.1, respectively) (Figure 4 and Table 3). These contaminant cells were likely to be responsible for the presence in culture of small clones with different morphology (Figure 4B, black arrowheads) that grew faster than CAFs and prevailed over the primary CAF culture (Figure 4C). These suboptimal results confirmed the importance of always double-checking both CD90.2 and CD45 expression, as well as cell morphology at the end of the enrichment process and during cell growth in culture.
Use of isolated cells to evaluate CAF-targeting potential of engineered nanodrugs 
The freshly isolated CAFs can be used for several applications ranging from basic research to pharmacological studies. This group&#8217;s aim is to develop HFn nanocages that can specifically target CAFs. HFn was functionalized with a specific anti-FAP antibody fragment (HFn-FAP) at two different protein:antibody ratios (a lower 1:1 and a higher 1:5), and their binding with CAFs was tested. As FAP was used as a surface biomarker of pro-tumorigenic CAFs, it was of fundamental importance to check FAP expression on isolated CAFs (Figure 3C). FAP expression was followed over 5 passages in culture to confirm that the primary CAF culture maintained its original characteristics.
FAP functionalization on HFn was found to contribute to a significant shift toward CAF targeting as compared to bare HFn, and the lower amount of antibody (1:1) was enough to observe this effect (Figure 5A). However, this was not observed with the tumor 4T1 cells used to set up the in vivo tumor model, wherein bare HFn showed higher binding than functionalized HFn (Figure 5B). This was most likely due to the absence of FAP overexpression in 4T1 cells and the preferential interaction of HFn with TfR1, which regulates HFn uptake in cells, as widely reported by this group27,33. These results confirm the usefulness of using primary cultures of breast CAFs to preliminarily screen the targeting capability of nanoparticles designed to tackle the tumor microenvironment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62504/62504fig01.jpg" /> 
Figure 1: Establishment of the 4T1 tumor model. Cells (105 cells/mouse) were injected into the mammary fat pad. Tumor growth was followed at days 5, 10, 15, 20, 25, and 30 after implantation (A) by measuring tumor volume with calipers and (B) by bioluminescence imaging. Tumor volumes and BLI are expressed as mm3 and counts, respectively. Results are reported as average &#177; SEM (n=6). (C) BLI representative images obtained 5, 10, 15, 20, 25, and 30 days after cell implantation confirm tumor growth until day 25, when it seems to reach a plateau. At the last time point of analysis (30 days), the BLI does not increase as compared to day 25. Starting at day 20, areas of necrosis and ulceration start to become visible in the central part of the tumor. Color Scale: Min = 1,194, Max = 20,462. Abbreviations: BLI = bioluminescence imaging; SEM = standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/62504/62504fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62504/62504fig02.jpg" /> 
Figure 2: Tumor sample preparation and flow cytometry characterization of isolated CAFs. Tumors were excised and reduced into (A) small pieces of approximately 1&#8211;2 mm with the help of a scalpel; (B) single cell suspension after tissue digestion and mechanical dissociation of an excised tumor; (C) cell pellet obtained after lysis of red blood cells; (D) flow cytometry analysis of CD45 and CD90.2 expression in cells obtained after removal of red blood cells and dead cells, (E) after depletion of non-cancer-associated fibroblasts, and (F) after enrichment of cancer-associated fibroblasts, where the majority of cells are CD90.2+ CD45- (blue rectangle). Abbreviations: CAFs= cancer-associated fibroblasts; CD = cluster of differentiation; PE-A = area of phycoerythrin; FITC-A = areas of fluorescein isothiocyanate. <a href="https://www.jove.com/files/ftp_upload/62504/62504fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62504/62504fig03.jpg" /> 
Figure 3: Morphological analysis of CAFs and FAP expression. (A, B) CAFs morphology was checked throughout all passages of culture by optical microscopy (different levels of confluence at passage 2 and passage 5) and compared to (D) 4T1 tumor cells; scale bars = 10 &#181;m. (C) Fibroblast activation protein (FAP) expression was evaluated by flow cytometry at the end of the isolation process on the collected CD90.2+ CD45- cells to confirm their molecular characteristics. A fluorescence threshold was set on unstained control cells (red graph) to quantify mean fluorescence intensity and the percentage of positive cells (FAP+) among antibody-stained cells (blue graph). Abbreviations: CAFs= cancer-associated fibroblasts; FAP = fibroblast activation protein; CD = cluster of differentiation; MFI = mean fluorescence intensity; Ab = antibody; FITC-A = area of fluorescein isothiocyanate; FAP+ = FAP-positive cells. <a href="https://www.jove.com/files/ftp_upload/62504/62504fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62504/62504fig04.jpg" /> 
Figure 4: Example of a sub-optimal CAFs isolation process. (A) Flow cytometry evaluation after the final isolation step revealed the presence of contaminant CD90.2- CD45+ and CD90.2- CD45- cells. (B) These cells can be seen as clones with small round morphology upon seeding (black arrowheads and inset in the bottom left corner of the panel) that (C) prevailed over CAFs after the third passage in culture. Scale bars = 10 &#181;m. Abbreviations: CAF = cancer-associated fibroblast; CD = cluster of differentiation; PE-A = area of phycoerythrin; FITC-A = areas of fluorescein isothiocyanate. <a href="https://www.jove.com/files/ftp_upload/62504/62504fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62504/62504fig05.jpg" /> 
Figure 5: Binding of HFn nanocages on CAFs and 4T1. HFn nanocages were fluorescently labeled with FITC, functionalized with an anti-FAP antibody fragment (HFn-FAP) at two different protein-antibody ratios (1:1 and 1:5), and incubated with (A) target CAFs and (B) 4T1 cells at 4 &#176;C for 2 h. Binding was evaluated by flow cytometry. (A) HFn-FAP binding with CAFs is significantly increased at both antibody fragment concentrations by three-fold as compared to bare HFn. (B) in contrast, a significantly higher binding of bare HFn was observed in 4T1 cells, where binding is not enhanced by FAP recognition. Results are reported as average &#177; standard deviation of three independent experiments. *** p = 0.0003; &#176;&#176; p = 0.0021 ; &#176;&#176;&#176; p = 0.0008. Abbreviations: CAFs= cancer-associated fibroblasts; FAP = fibroblast activation protein; HFn = recombinant variant of human ferritin heavy chain used as bare nanoparticle or conjugated; HFN-FAP = HFn nanoparticles functionalized with the variable portion of an anti-FAP antibody prepared at two HFn:Fab@FAP molar ratios, 1:1 and 1:5. <a href="https://www.jove.com/files/ftp_upload/62504/62504fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Cell Number&#160; (average &#177; SD)</td>
			<td>Extraction Yield Total (per passage)</td>
		</tr>
		<tr>
			<td>Excised tumor (Post red blood cell removal)</td>
			<td>1.27 x 108 &#177; 9.81 x 107</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>&#160;Excised tumor (Post dead cell removal)</td>
			<td>3.01 x 106 &#177; 9.61 x 105</td>
			<td>2.38%</td>
		</tr>
		<tr>
			<td>Post depletion cocktail</td>
			<td>1.15 x 105 &#177; 4.95 x 104</td>
			<td>0.11% (3.82%)</td>
		</tr>
		<tr>
			<td>Post enrichment</td>
			<td>3.00 x 104 &#177; 1.2 x 104</td>
			<td>0.027% (26.09%)</td>
		</tr>
	</tbody>
</table>
Table 1: Total cell count after each step of isolation of cancer-associated fibroblasts.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Distribution (%)</td>
			<td>CD90.2+ CD45-</td>
			<td>CD90.2+ CD45+</td>
			<td>CD90.2- CD45+</td>
			<td>CD90.2- CD45-</td>
		</tr>
		<tr>
			<td>Excised tumor (Post dead cell removal)</td>
			<td>1.81 &#177; 0.98</td>
			<td>10.78 &#177; 4.51</td>
			<td>56.57 &#177; 14.05</td>
			<td>28.20 &#177; 17.57</td>
		</tr>
		<tr>
			<td>Post depletion cocktail</td>
			<td>69.33 &#177; 16.75</td>
			<td>0.14 &#177; 0.13</td>
			<td>0.55 &#177; 0.63</td>
			<td>39.89 &#177; 30.31</td>
		</tr>
		<tr>
			<td>Post enrichment</td>
			<td>93.14 &#177; 3.3</td>
			<td>0.09&#160; &#177; 0.1</td>
			<td>&#160;0.09 &#177; 0.08</td>
			<td>6.69 &#177; 3.4</td>
		</tr>
	</tbody>
</table>
Table 2: Cell distribution according to expression of CD90.2 and CD45 after each passage of isolation of cancer-associated fibroblasts.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Distribution (%)</td>
			<td>CD90.2+ CD45-</td>
			<td>CD90.2+ CD45+</td>
			<td>CD90.2- CD45+</td>
			<td>CD90.2- CD45-</td>
		</tr>
		<tr>
			<td>Post Enrichment (no pooling)</td>
			<td>75.80 &#177; 2.9</td>
			<td>1.49 &#177; 0.4&#160;</td>
			<td>5.97 &#177; 1.5</td>
			<td>16.75 &#177; 1.1</td>
		</tr>
	</tbody>
</table>
Table 3: CD90.2 and CD45 expression of cells collected after a sub-optimal cancer-associated fibroblast isolation experiment.

Watch this Scientific Journal Video about Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles at JoVE.com

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

isolation-primary-cancer-associated-fibroblasts-from-syngeneic-murine

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Cancer Research

5.3K Views.  Istituti Clinici Scientifici Maugeri IRCCS. This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment. 

Video: Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.