Name: testing cancer immunotherapeutics in a humanized mouse model bearing human tumors
Uploaded: 2022-12-16T13:50:00
Description: Watch this Scientific Journal Video about Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors at JoVE.com

We would like to thank both the Animal Research Facility (OLAR) for their care of our mice, and the Flow Cytometry Shared Resource supported by the Cancer Center Support Grant (P30CA046934) at our institute for their immense help in all our work. We also acknowledge both Gail Eckhardt and Anna Capasso for our inaugural collaborations studying immunotherapies to human PDXs in our HIS-BRGS model.&#160;This study was supported in part by the National Institutes of Health P30CA06934 Cancer Center Support Grant with use of the PHISM (Pre-clinical Human Immune System Mouse Models) Shared Resource, RRID: SCR_021990 and Flow Cytometry Shared Resource, RRID: SCR_022035.&#160;This research was supported in part by the NIAID of the National Institutes of Health under Contract Number 75N93020C00058.

All animal work was performed under animal protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC Protocols #00593 and #00021). All animal work was performed in accordance with the Office of Laboratory Animal Resources (OLAR), an accredited facility by the American Association for Laboratory Animal Care, at the University of Colorado Denver Anschutz Medical Campus. All human cord blood samples were obtained as donations from de-identified donors and are thus not subject...

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Over the past 6 years, using our expertise in both immunology and humanized mice, our research team has developed a much needed preclinical model to test immunotherapies on a variety of human tumors3,7,30,31. This protocol emphasizes the consideration of the variability of the model, with special attention to the immunotherapy-centric human T cell populations. In this protocol, the generation o...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors</a>. The Authors section was updated from:
Jordi M. Lanis1 
Matthew S. Lewis1 
Hannah Strassburger1 
Stacey M. Bagby2 
Adrian T. A. Dominguez2 
Juan A. Mar&#237;n-Jim&#233;nez3 
Roberta Pelanda1 
Todd M. Pitts2 
Julie Lang1 
1Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
2Division of Oncology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
3Department of Medical Oncology, Catalan Institute of Oncology (ICO-L&#8217;Hospitalet)
to:
Jordi M. Lanis1 
Matthew S. Lewis1 
Hannah Strassburger1 
Kristina Larsen1 
Stacey M. Bagby2 
Adrian T. A. Dominguez2 
Juan A. Mar&#237;n-Jim&#233;nez3 
Roberta Pelanda1 
Todd M. Pitts2 
Julie Lang1 
1Department of Immunology and Microbiology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
2Division of Oncology, School of Medicine, University of Colorado Denver Anschutz Medical Campus 
3Department of Medical Oncology, Catalan Institute of Oncology (ICO-L&#8217;Hospitalet)

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors</a>. The Authors section was updated.

Erratum: Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Mouse cancer models are important for establishing basic mechanisms of tumor growth and immune escape. However, cancer treatment studies in mouse models have yielded finite translation to the clinic due to limited syngeneic models and species-specific differences1,2. The emergence of immune therapies as a dominant approach to control tumors has reiterated the need for an in vivo model with a functional human immune system. Advancements in human immune system mice (HIS mice) over the past decade have made it possible to study immuno-oncology in vivo in a wide variety of cancer types and immunotherapeutic agents3,4,5,6. Human tumor models, including cell-line derived and patient-derived xenografts (CDX and PDX, respectively), grow well in HIS mice and in most cases are nearly identical to their growth in the immunodeficient host lacking human hematopoietic engraftment7,8. Based on this key finding, researchers have been using the HIS mouse model to study human immunotherapies, including combination therapies designed to alter the tumor microenvironment (TME) to decrease immunosuppression and thus enhance immune-directed tumor killing. These preclinical models help address the issues of heterogeneity of human cancers, and can also predict treatment success as well as monitor immune related drug toxicities9,10.
The production of a mouse model with a human immune system through the introduction of human hematopoietic stem cells requires a recipient immunodeficient mouse that will not reject the xenograft. Current HIS mouse models are derived from immunodeficient mouse strains that were reported over 30 years ago. The first immunodeficient mouse strain described was SCID mice that lacked T and B cells11, followed by a hybrid NOD-SCID with an SIRP&#945;&#160;polymorphism responsible for mouse macrophage tolerance to human cells, due to increased binding for the NOD SIRP&#945; allele to the human CD47 molecule12,13. In the early 2000s, the deletion of the common gamma chain of the IL-2 receptor (IL-2R&#947;c) on both BALB/c and NOD immunodeficient strains was a game changer for enhanced human engraftment, due to genetic deletions forbidding host NK cell development14,15,16,17. Alternative models, such as BRG and NRG mice, achieve T and B cell deficiency through deletion of the Rag1 or Rag2 gene, required for T and B cell receptor gene rearrangements and thus the maturation and survival of lymphocytes18,19. The BRGS (BALB/c -Rag2nullIl2R&#947;CnullSirp&#945;NOD) mouse used herein combines the IL-2R&#947; chain deficiency and the NOD SIRP&#945; allele on the Rag2-/- background, resulting in a highly immunodeficient mouse without T, B, or NK cells, yet with sufficient vigor and health to allow for long term engraftment of more than 30 weeks13.
HIS mice can be generated in multiple ways, with human PBMC injection being the most direct method15,18,20. However, these mice have a pronounced expansion of activated human T cells that results in graft versus host disease (GVHD) by 12 weeks of age, preventing long-term studies. Alternatively, human hematopoietic stem cells from umbilical cord blood (CB), bone marrow, and fetal liver can also be used for engraftment and production of the human immune system de novo. In this system, the hematopoietic stem cells produce a multi-lineage human immune system with the generation of T, B, and innate immune cells that are importantly tolerant of the mouse host, compared to the PBMC mice that develop mostly T cells. Therefore, GVHD is absent or greatly delayed, and studies can be extended to mice up to 10 months of age. CB provides an easy, accessible, and noninvasive source of CD34+ human hematopoietic stem cells that facilitates the engraftment of multiple HIS mice with genetically identical immune systems17,18,20,21. Over the past few years, HIS mouse models have been used extensively to study immunotherapy and the TME3,4,5,6. Despite the development of human derived immune systems in these mice, human xenograft tumors grow at similar rates compared to the control immunodeficient mice and allow for the complex interplay between the cancer cells and immune cells, which is important for maintaining the microenvironment of the engrafted PDX3,7,8. This protocol has been used to perform over 50 studies testing treatments in HIS-BRGS mice with PDXs and CDXs. An important conclusion is that human tumors in the HIS mice maintain their unique TME as defined by molecular evaluation of the tumor relative to the initial patient sample and immune infiltrate characteristics3,22,23. Our group focuses on in-depth evaluation of the HIS in both immune organs and the tumor using multi-parameter flow cytometry. Herein, we describe a protocol for the humanization of BRGS mice, evaluation of chimerism, implantation of human tumors, tumor growth measurements, cancer treatment administration, and analysis of the HIS cells by flow cytometry.

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		<tr>
			<td>1 mL syringe w/needles</td>
			<td>McKesson</td>
			<td>1031815</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Grenier Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Grenier Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi</td>
			<td>130-092-545</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Golgi Stop Protein Transport Inhibitor with monensin</td>
			<td>BD Bioscience</td>
			<td>BDB563792</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP1600100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Stim Cocktail</td>
			<td>Life Technologies</td>
			<td>509305</td>
			<td></td>
		</tr>
		<tr>
			<td>Chill 15 Rack</td>
			<td>Miltenyi</td>
			<td>130-092-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton-plugged glass pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Basement membrane extract</td>
			<td>R&#38;D Systems</td>
			<td>363200502</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytek Aurora</td>
			<td>Cytek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma</td>
			<td>9003-98-9</td>
			<td></td>
		</tr>
		<tr>
			<td>eBioscience FoxP3/Transcription Factor Staining Buffer Set</td>
			<td>Invitrogen</td>
			<td>00-5523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Embryonic Stemcell FCS</td>
			<td>Gibco</td>
			<td>10439001</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes; 1.5 mL volume</td>
			<td>Grenier Bio-One</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Benchmark</td>
			<td>100-106 500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Hypaque</td>
			<td>GE Healthcare</td>
			<td>45001752</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - fine</td>
			<td>Roboz Surgical&#160;</td>
			<td>RS5045</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps normal</td>
			<td>Dumont</td>
			<td>RS4919</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>F75P1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Glass Slides</td>
			<td>Corning</td>
			<td>1255310</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tubes</td>
			<td>Miltenyi&#160;&#160;&#160;</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>GentleMACS Dissociator</td>
			<td>Miltenyi</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>glass pipettes</td>
			<td>DWK Life Sciences</td>
			<td>63A53</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS w/ Ca &#38; Mg</td>
			<td>Sigma</td>
			<td>55037C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Corning</td>
			<td>MT25060CI</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG standard</td>
			<td>Sigma</td>
			<td>I2511</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM standard</td>
			<td>Sigma</td>
			<td>401108</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase DL</td>
			<td>Roche</td>
			<td>5466202001</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue</td>
			<td>Thermo</td>
			<td>L23105</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td>1140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human IgG-AP</td>
			<td>Southern Biotech</td>
			<td>JDC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human IgG-unabeled</td>
			<td>Southern Biotech</td>
			<td>H2</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human IgM-AP</td>
			<td>Southern Biotech</td>
			<td>UHB</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human IgM-unlabeled</td>
			<td>Southern Biotech</td>
			<td>SA-DA4</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiRad 350</td>
			<td>Precision X-Ray</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>45000-446</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>p-nitrophenyl substrate</td>
			<td>Thermo</td>
			<td>34045</td>
			<td></td>
		</tr>
		<tr>
			<td>PRISM</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu FLT3L</td>
			<td>R&#38;D systems</td>
			<td>308-FK-005/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu IL6</td>
			<td>R&#38;D systems</td>
			<td>206-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec Hu SCF</td>
			<td>R&#38;D systems</td>
			<td>255SC010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>45000-39</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>8047-15-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>McKesson</td>
			<td>862945</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes 25 mL</td>
			<td>Fisher Scientific</td>
			<td>1367811</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filter</td>
			<td>Nalgene</td>
			<td>567-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile molecular water</td>
			<td>Sigma</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeti Cell Analyzer</td>
			<td>Bio-Rad</td>
			<td>12004279</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Green</td>
			<td>biolegend</td>
			<td>423112</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing cancer immunotherapeutics in a humanized mouse model bearing human tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Following the flank tumor protocol and experimental timeline (Figure 1), the tumor growth and immune response to a targeted tyrosine kinase inhibitor (TKI) therapy and nivolumab combination treatment was studied in two distinct human colorectal cancer (CRC) PDXs. The TKI drugs have been studied in immunodeficient hosts to evaluate tumor growth only29. This model enabled the study of changes in the immune response of the TKI alone, and more importantly, in combination ...

Watch this Scientific Journal Video about Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors at JoVE.com

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

This protocol outlines the generation of Human Immune System, or HIS mice for preclinical immuno-oncology studies. Mouse cancer models are limited in their diversity and translate poorly to the clinic. This technique serves as a preclinical model for immunotherapy studies by evaluating the treatment of human tumors in a mouse with a human immune system.The humanized mouse model can be used to study multiple aspects of human immunology, including responses to many infections, or cancers of the human immune system. Acquiring a reliable and robust source of cord blood and maintaining the health of the immunodeficient mouse colony can be challenging. Expertise in flow cytometry and immunological analysis is essential.Demonstrating the procedure will be Kristina Larsen, a research services professional from my laboratory. To begin, resuspend the CD34-positive cell pellet isolated from cord blood in 300 microliters of magnetic cell separator buffer per one times 10 to the eighth cells. Add 100 microliters of FCR blocking reagent per one times 10 to the eighth cells, followed by 100 microliters of CD34-positive magnetic beads per one times 10 to the eighth cells, and incubate at four degrees Celsius for 30 minutes.Next, add five milliliters of magnetic cell separator buffer per one times 10 to the eighth cells, and spin at 360 g for 10 minutes at four degrees Celsius. Repeat the wash step and resuspend the pellet in 500 microliters per 100 million cells of magnetic cell separator buffer in a 15 milliliter conical tube labeled unfractionated. Label two more 15 milliliter tubes CD34-negative and CD34-positive Place the three tubes on a cooling rack.Separate the cells using the two column positive selection program on an automatic magnetic cell separator in a biosafety cabinet. Next, for expanding and freezing CD34-positive human stem cells, prepare cord blood, or CB medium as described in the manuscript, and pass through a 0.22 micron filter. Resuspend the CD34-positive cells at 100, 000 per milliliter of CB medium, and incubate at 37 degrees Celsius.On day three, add an equal volume of CB medium without cytokines to the flask. On day five, harvest the expanded CD34-positive cells. Pipette the cell suspension up and down, and collect in a 50 milliliter conical tube.Add enough CB medium to cover the bottom of the flask. Using a cell scraper, scrape the entire bottom of the flask. Collect all the media into the same 50 milliliter tube, and centrifuge.Resuspend the cells in two milliliters of CB medium, and centrifuge. Aspirate the medium down to the pellet, and resuspend the pellet in a freezing medium. Next, begin CD34-positive cell preparation three hours post-pup irradiation.Warm 10 milliliters of CB media in a 50 milliliter tube. Retrieve one vial of in vitro expanded and frozen CD34-positive cells for every four to six pups to inject. Rapidly thaw at 50 to 55 degrees Celsius until just a small amount of ice is visible, and add the cells to the warmed CB medium.Use one milliliter of medium to rinse each vial, and spin the cells at 360 g for 12 minutes at four degrees Celsius. Aspirate the medium carefully. Resuspend the pellet in two milliliters of CB media and count the cells.Again, centrifuge the cells, aspirate the medium, and resuspend the pellet in 100 microliters of sterile PBS per n plus one pups to inject, resulting in 250, 000 to 450, 000 CD34-positive cells per mouse. For PBMC isolation from mouse blood, mix the blood heparin by gently pipetting up and down, and slowly overlay on top of 500 microliters of 1.077 gram per milliliter density gradient being careful not to disturb the interface. Centrifuge the tube at 1, 220 g for 20 minutes at room temperature with no breaks.Remove as many cells as possible from the buffy coat with a 200 microliter pipette, and add to the new 1.5 milliliter tube containing 750 microliters of harvest medium. Centrifuge at 360 g for 11 minutes. Aspirate the medium to 50 microliters, and resuspend the pellet in 750 microliters of harvest medium.Acquire the data for 100 microliters of each sample on a flow cytometer. Then import the fcs file to the flow data editing software, and apply a polygon gate to an FSC-A versus SSC-A plot. Change the axes to FSC-A versus FSC-H, and gate the cells on the linear diagonal, excluding the doublets that protrude from the line.Select these cells and change the axes to hCD45 versus mCD45. Apply a polygon gate to the hCD45-positive population, and apply the name human. Apply a polygon gate to the mCD45-positive population, and apply the name mouse.Create a counting statistic for human and mouse populations. Next, select the human population, and change the axes to CD19 versus CD3. Apply a polygon gate to the CD19-positive cells, and name them B cells.Apply a polygon gate to the CD3-positive population, and name it T-cells. Then apply a polygon gate to the double negative population, and name it non-TB. Select the T-cell population and change the axes to CD4 versus CD3.Apply a polygon gate to the CD4-positive population, and name it CD4-positive. Apply a polygon gate to the CD4-negative population, and name it CD8. Select the non-TB population, and change the axes to CD56 versus myeloid.Apply a polygon gate to the total CD56-positive population, and name it NK cells. Then apply a polygon gate to the CD56-negative and myeloid-positive population, and name it myeloid. Create a table for the percentage and count statistics of all populations, and export it to a spreadsheet.Calculate percent hCD45 chimerism using the indicated formula. After growing tumor cells in the mouse and administering drug treatments, harvest the tissues. Place the mouse on a foam dissection board with pins to hold them in place and the arms and legs extended at 45 degrees.Make an incision up the middle of the torso starting near the pelvis and extending to the chin. Pull the skin to the edge, and hold it in place with pins. Extract the lymph nodes using fine forceps in the order axillary, cervical, mesenteric, inguinal, and hiatal.Place the lymph nodes on one side of the frosted glass slide in a Petri dish in eight milliliters of harvest medium. Holding the slides at perpendicular angles with the frosted edges inward, gently press the tissues until the cellular contents are released. Rinse the slide several times by pulling them apart and together to release the maximal number of cells.Collect the cells with a five inch glass pipette, and filter them through a nine inch cotton-plugged pipette into a labeled 15 milliliter conical tube. Next, extract the tumor from the open flank by holding the tumor with forceps while slowly snipping at the tumor margins with dissection scissors. Weigh the tumor and remove 1/4 of the tumor for RNA and immunohistochemistry processing.Place the remaining 3/4 of the tumor into a six centimeter dish, and mince it into one millimeter pieces using a scalpel blade. Transfer the tumor pieces into a dissociation tube. Then rinse the dish with incomplete TIL medium, and add it to the tube.Add collagenase preparation, and dissociate the tissue using mechanical dissociation at 37 degrees Celsius for 30 minutes to one hour. Pass the suspension over a 100 micron filter into a 50 milliliter conical tube and rinse the filter with 10 milliliters of TIL complete media. Centrifuge and resuspend the pellet in just enough harvest medium with DNase, so that the cells suspension can easily pass through a P1000 pipette tip and record the volume for downstream analysis.In PDX CRC 307P, the combination treatment slowed the growth of the tumor, as determined by tumor growth volumes over time, tumor weights, and the specific growth rate. PDX CRC 307M tested in a different cohort of mice was less affected by the same combination treatment in HIS BRGS mice. Based on overall human hCD45-positive and human T-cell hCD3-positive chimerism in the blood before tumor implantation, both parameters increased in the peripheral immune system and the TIL in combination with treated CRC 307P-bearing mice, but not the CRC 307M model.Investigation of the human T-cells revealed more activated T-cells in the CRC 307P tumors, but not the lymph from combination treated mice. Also, there were more effector memory CD8-positive T-cells and fewer TIM-3-positive T-cells in the combination treated CRC 307P tumors. In contrast, this difference was not noted in the CRC 307M model.Further, no changes in the frequencies of cytotoxic T-cell populations were observed among the combination treated mice, although higher cytotoxic T-cells were observed in untreated. Combination treatment did not affect the frequencies of Tregs in either CRC 307P lymph organs, or tumors, although the CRC 307M data showed a trend of reduced Tregs. Immune related changes in tumor cells were elevated using flow cytometry.Increased expression of MHC class I and class II was observed on the CRC 307P tumor cells excised from combination treated HIS BRGS mice. In the CRC 307M model, the same drug treatment induced HLA class II expression on the tumor cells. Similarly, the combination treatment resulted in increased PD-L1 expression on the EpCAM-positive CRC 307P tumor cells.Finally, correlations of immune responses with tumor growth were investigated. Increased CD4-positive T-cells showed a significant correlation with smaller tumor growth, and more specifically the HLA-DR-positive activated T-cells in the combination treated HIS mice. Sterile technique while isolating the CD34-positive cells, as the mice are extremely immunodeficient.The resulting chimerism is variable within and between cord blood donors, and T-cells have the largest variation. Unlimited immunotherapy combinations can be performed, as well as mechanistic and drug dosing kinetic studies, for example, deletion of CD8 T-cells to confirm their role. Importantly, drug related toxicities can also be studied.It provided a more relevant and accessible preclinical model to test human immunotherapies for translation into the clinic. Several clinical trials are in progress now based on these data.

testing-cancer-immunotherapeutics-humanized-mouse-model-bearing-human

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...