Name: portal vein injection of colorectal cancer organoids to study the liver metastasis stroma
Uploaded: 2021-09-03T13:45:00
Description: Watch this Scientific Journal Video about Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma at JoVE.com

This study was supported by grants from the National Health and Medical Research Council (APP1156391 to D.L.W., S.L.W.) (APP1081852 to D.L.W., APP1140236 to S.L.W., APP1099283 to D.L.W.,); Cancer Council SA Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health (MCF0418 to S.L.W., D.L.W.); a Grant-in-Aid for Scientific Research (B) (20H03467 to M.T.) commissioned by the Ministry of Education, Culture, Sports, Science and Technology of Japan; AMED-CREST (Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (19gm0810007h0104 and 19gm1210008s0101 to A.E.); the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from AMED (19cm0106332h0002 to A.E.); Japan Society for the Promotion of Science Overseas Challenge Program for Young Researchers (to H.K.), Takeda Science Foundation Fellowship (to H.K.), Greaton International Ph.D. Scholarship (to H.K.), Lions Medical Research Foundation Scholarship (to K.G.).
We thank Dr. Leszek Lisowski at Vector and Genome Engineering Facility (VGEF), Children&#39;s Medical Research Institute (CMRI) (NSW, AUSTRALIA) for producing recombinant AAV vectors.

All animal procedures in this article were reviewed and approved by the South Australian Health and Medical Research Institute Animal Ethics Committee (Approval number, SAM322).
1. Tail vein injection of adeno-associated virus
NOTE: Adeno-associated virus (AAV) should be handled as a biohazard under Biosafety Level 1 guidelines. Please refer to the published protocol for AAV preparation, purification, and titration33. Hepatocyte-tropic AAV, AAV...

In this study, we have shown that portal vein injection of mouse CRC organoids reproducibly generates fibroblast-rich liver metastases that mimic histological features of human CRC hepatic metastases. Furthermore, when combined with stroma-directed therapeutics such as AAV8-mediated gene therapy, this preclinical model serves as a useful tool to assess therapeutic effects on mouse survival and tumor growth.
There are, at least, two critical steps in the protocol. Firstly, it is important to pr...

Colorectal cancer (CRC) is a major cause of cancer mortality worldwide1. More than half of the CRC patients develop hepatic metastasis that occurs through the portal vein dissemination1. Currently, there are no effective therapeutics that can cure advanced liver metastasis, and most patients succumb to metastatic disease.
The metastatic niche or tumor microenvironment plays a key role in engraftment and growth of disseminated CRC cells2. Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, promote or restrain cancer progression through secreting growth factors, remodeling the extracellular matrix (ECM), and modulating immune landscapes and angiogenesis3,4,5. CAFs also confer resistance to chemotherapies and immunotherapies3. Moreover, CAFs regulate initiation and progression of CRC liver metastasis and predict prognosis in patients with CRC3,6,7,8. Thus, CAF-related factors could be exploited for the development of therapeutic strategies to inhibit CRC liver metastasis. However, the lack of satisfactory mouse models to study the metastatic tumor stroma has been a major obstacle to developing stroma-targeted therapies.
Currently, animal models to study CRC liver metastasis include primary CRC models that spontaneously develop hepatic metastasis and cancer cell transplantation models into the liver. Primary CRC mouse models, such as genetically engineered mouse models and colonic injection of cancer cells, rarely show metastasis to the liver9,10,11,12. Moreover, even if a liver metastasis is observed, these models show long latency from the primary tumor induction to metastasis, and potentially die of primary tumor burden12. To efficiently generate CRC liver metastases, cultured CRC cells are transplanted into the liver&#160;using three injection approaches: intra-splenic injection, direct intra-parenchymal injection into the liver, and portal vein injection. Intra-splenically injected cancer cells spread into the splenic vein, the portal vein, and ultimately to the liver13,14. However, the intra-splenic injection yields a lower tumor take ratio compared with other transplantation models15,16. With intra-splenic injection, surgical removal of the spleen is performed to avoid cancer growth in the spleen, which can potentially compromise immune cell maturation17. Furthermore, intra-splenic injection can also result in unintended tumor growth in the spleen and abdominal cavity18, complicating liver metastasis analyses. Direct intra-parenchymal injection into the liver efficiently induces hepatic metastasis16,19,20. Nonetheless, this approach does not fully recapitulate a biological step of liver metastasis that naturally occurs through portal vein dissemination. Using direct injection into the liver, entry of cancer cells into a non-portal, but systemic circulation can also result in multiple large lung metastases16. Although a majority of patients with CRC liver metastasis show multiple tumor nodules in the liver21, direct injection into a specific liver lobe generates a single tumor mass19,20. Portal vein injection or mesenteric vein injection, though technically challenging, allows efficient delivery of tumor cells into the liver in a manner that recapitulates the growth patterns seen in patients17. This strategy can minimize the possibility of secondary-site metastases and enables rapid growth of cancer cells in the liver, simplifying mouse survival analyses.
Historically, colorectal cancer cell lines such as mouse MC-38, human HT-29, and SW-620 were used to generate mouse models of hepatic metastasis22,23. However, these colorectal cancer cell lines do not induce a desmoplastic stromal reaction. Low stromal content in the tumors makes it difficult to investigate the biological roles of cancer-associated fibroblasts. Recent advances in CRC organoids and their transplantation have offered useful platforms to assess vital roles of the stroma in cancer progression24. Liver transplantation of CRC organoids generates a fibroblast-rich tumor microenvironment and has provided novel insights into stromal research6,25. Currently, portal or mesenteric vein injection of organoids has become a gold standard approach to generate CRC liver metastasis6,25,26,27,28. Nonetheless, to our knowledge, no previous papers have described detailed methods for the portal vein injection of colorectal tumoroids. Here, we present a methodology for using portal vein injection of CRC organoids to develop novel adeno-associated virus (AAV)-mediated stroma-directed therapy.
Hepatocytes are an important constituent of the metastatic tumor microenvironment in the liver and play a critical role in metastatic cancer progression29. Inspired by the success of AAV gene therapy approaches to induce protein expression in hepatocytes in non-neoplastic patients30,31, we investigated a similar approach but aimed at modifying the liver tumor microenvironment in CRC25. As such, we also describe herein the tail vein injection of AAV8 to induce expression of anti-tumorigenic proteins to modify the liver tumor microenvironment. The AAV8 serotype, designated by the choice of viral capsid protein during virus production, leads to high transduction efficiency specifically of hepatocytes (i.e., targeted gene expression in the liver tumor microenvironment)32. We have previously shown that Islr (immunoglobulin superfamily containing leucine-rich repeat) is a CAF-specific gene that induces bone morphogenetic protein (BMP) signaling, reduces CRC tumoroid growth, and promotes Lgr5+ intestinal stem cell differentiation25. We tested whether AAV8-mediated overexpression of the cancer-restraining stromal gene, Islr, in hepatocytes could attenuate hepatic metastasis progression by performing portal vein injection of CRC tumoroids in AAV8-Islr-treated mice.
In this paper, we first describe the tail vein injection procedure of liver tropic AAV. Then, we describe a method for tumoroid cell preparation and portal vein injection into the AAV-treated mice. Finally, we present approaches to monitor metastatic tumor progression to assess the efficacy of stroma-directed therapeutics.

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			<td>10% Formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
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		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
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		<tr>
			<td>33-gauge needle</td>
			<td>TSK</td>
			<td>LDS-33013</td>
			<td>For portal vein injection</td>
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		<tr>
			<td>4-0 vicryl suture</td>
			<td>ETHICON</td>
			<td>J494G</td>
			<td></td>
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			<td>40-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
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			<td>5 mL Syringe</td>
			<td>BD</td>
			<td>302130</td>
			<td>Used to apply saline to the intestine after portal vein injection</td>
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			<td>50 mL centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
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			<td>50 mL syringe</td>
			<td>TERUMO</td>
			<td>SS*50LE</td>
			<td>Luer lock syringe for perfusion fixation</td>
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			<td>70% Isopropyl alcohol wipe</td>
			<td>Briemar</td>
			<td>5730</td>
			<td></td>
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		<tr>
			<td>Anaesthesia machine</td>
			<td>Darvall</td>
			<td>9356</td>
			<td></td>
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		<tr>
			<td>&#945;SMA antibody</td>
			<td>DAKO</td>
			<td>M0851</td>
			<td>Clone 1A4. 1/500 dilution for immunohistochemistry</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>TROY</td>
			<td>N/A</td>
			<td>ilium Temvet Injection, 300 &#181;g/ml Buprenorphine</td>
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		<tr>
			<td>Cotton buds</td>
			<td>Johnson &#38; Johnson</td>
			<td>N/A</td>
			<td>Johnson&#39;s pure cotton bud applicators. Need to be autoclaved before use.</td>
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			<td>D-luciferin</td>
			<td>Biosynth</td>
			<td>L-8220</td>
			<td></td>
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		<tr>
			<td>Electric shaver</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>HAMILTON</td>
			<td>81020</td>
			<td>For portal vein injection</td>
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			<td>Heat box (animal warming chamber)</td>
			<td>Datesand</td>
			<td>MK3</td>
			<td></td>
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			<td>Heat lamp</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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			<td>Hemostatic sponge</td>
			<td>Pfizer</td>
			<td>09-0891-04-015</td>
			<td>Gelfoam absorbable gelatin sponge, USP, 12-7 mm</td>
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		<tr>
			<td>India ink</td>
			<td>Talens</td>
			<td>44727000</td>
			<td></td>
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		<tr>
			<td>Injection syringe and needle</td>
			<td>BD</td>
			<td>326769</td>
			<td>For tail vein injection</td>
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		<tr>
			<td>Islr probe (RNAscope)</td>
			<td>ACD</td>
			<td>450041</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>988-3244</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image Software</td>
			<td>Perkin Elmer</td>
			<td>128113</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>MRI fibrosis tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/MRI_Fibrosis_Tool</td>
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		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope kit</td>
			<td>ACD</td>
			<td>322300</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent restrainer</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
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		<tr>
			<td>Rosa26-Cas9 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>024858</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Pfizer</td>
			<td>PHA19042010</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td>Sold by multiple suppliers</td>
		</tr>
		<tr>
			<td>Skin staplers</td>
			<td>Able Scientific</td>
			<td>AS59028</td>
			<td>9 mm wound clips</td>
		</tr>
		<tr>
			<td>Stapler applicator</td>
			<td>Able Scientific</td>
			<td>AS59026</td>
			<td>9 mm wound clip applicator</td>
		</tr>
		<tr>
			<td>Stapler remover</td>
			<td>Able Scientific</td>
			<td>AS59037</td>
			<td>Wound clip remover</td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>Multigate</td>
			<td>29-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gauze</td>
			<td>Sentry Medical</td>
			<td>GS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical anesthesia cream</td>
			<td>EMLA</td>
			<td>N/A</td>
			<td>EMLA 5% cream, 25 mg/g lignocaine and 25 mg/g prilocaine</td>
		</tr>
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			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td>Recombinant cell-dissociation enzyme mix</td>
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		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
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</table>

portal vein injection of colorectal cancer organoids to study the liver metastasis stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

To induce AAV-mediated overexpression of a tumor-restraining stromal gene, Islr4,25,43,44, in hepatocytes, we intravenously injected Islr-encoding AAV8. 1.0 x 1011 viral genomes (vg) of AAV8-Islr, or as a control, AAV8-mRuby2, was injected into the adult mouse tail vein (Figure 1A). Two weeks after the tail vein inject...

Watch this Scientific Journal Video about Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma at JoVE.com

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

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