Author:

Sheri Barnes, PhD | Director, Scientific Development

Date:

December 2017

Colorectal cancer is the fourth most common cancer diagnosed in the United States. Colorectal cancer represents the third leading cause of cancer-related deaths in women and the second in men. In 2017, over 135,000 estimated new cases of colorectal cancer in the United States will be diagnosed and more than 50,000 patient deaths will occur. Prevention and early detection initiatives over the last several decades together with improved treatment options have resulted in reductions in colorectal cancer diagnoses and deaths. These measures have also increased the five-year overall survival rate to 64.9%, but survival drops precipitously for those patients whose cancer is not detected early.1 For this reason, the development of new treatments for colorectal cancer is a continual need. In this model spotlight, MI Bioresearch will be highlighting the colon adenocarcinoma model, MC38, and how it supports the development of immuno-oncology treatments.

The advent of immunotherapy has necessitated syngeneic mouse tumor models with desirable growth kinetics and response to immunomodulatory agents to further advance development of immuno-oncology treatments. One of the colon adenocarcinoma models, MC38, has been characterized by MI Bioresearch to support development of these agents. MC38 was isolated from a colon tumor in a C57BL/6 mouse following long term exposure to the carcinogen DMH (1,2-dimethylhydrazine dihydrochloride).2 As described below, MC38 has a favorable response profile to immunomodulatory antibodies, and evidence via flow cytometry suggests its tumor microenvironment is amenable to immune activation. Taken together, MC38 is well positioned to be a powerful immuno-oncology model with significant utility in drug development.

The doubling time of MC38 is ~4 days, a moderate growth rate which can facilitate up to a three-week dosing window for test agents to elicit their anti-tumor activity. Figure 1 demonstrates mean (1A) and individual (1B-E) growth of control tumors compared to those treated with focal radiation or checkpoint inhibitors anti-mPD-1 or anti-mPD-L1.  Dosing with all test agents began once tumors were established (~100mm3). While the response of anti-mPD-L1 was modest, increasing the time to evaluation size (TES) by 7.5 days, the response of radiation and anti-mPD-1 were marked, with TES extensions of 19.6 and 14.7 days, respectively.  The clear effect of these treatments can allow for additive or synergistic improvement in combination with candidate molecules.

Fig. 1: Mean and individual growth of MC38 tumors following radiation or checkpoint inhibitor therapy.
Fig. 1: Mean and individual growth of MC38 tumors following radiation or checkpoint inhibitor therapy.

In contrast, the response of MC38 to the costimulatory molecules anti-mCD137, anti-mGITR, and anti-mOX40 was much less robust (Figure 2A and 2B-E) compared to responses illustrated in Figure 1. Tumor growth delay with these treatments extends only 1.4 to 6.9 days beyond control. However, intimations of activity demonstrating room for improvement are present with these agents as well, making MC38 an attractive model for combination therapy with a wide range of immunomodulatory agents.

Fig. 2: Mean and individual growth of MC38 tumors following costimulatory antibody treatment.
Fig. 2: Mean and individual growth of MC38 tumors following costimulatory antibody treatment.

Equally important to treatment response is the immune composition of syngeneic tumors. To learn more about the immune infiltrate of these MC38 tumors, we analyzed T cell and myeloid cell populations of naive tumors in the log phase of growth. The gating strategies for T cell (Figure 3A) and myeloid cell (Figure 3B) populations are illustrated below and results are listed in Table 1. While some variability exists across animals as one would expect, the trends across subsets are consistent. These tumors exhibit a low infiltrate of CD45+ cells, but overall a favorable T cell profile with populations of CD8+ T cells making up 25-50% of CD3+ cells.  Likewise, the Treg population is relatively small resulting in an advantageous CD8+/Treg ratio between 2.9 and 6.1. In contrast, the presence of myeloid cell populations was modest in the tumors tested. Overall, the percentages of MDSC and macrophages present in these tumors are lower compared to other syngeneic models such as 4T1, a well characterized syngeneic breast model with high myeloid cell content.

Gating Strategies for T Cell and Myeloid Cell Panels Based on Total Live Cells

Fig. 3A: T Cell Populations
Fig. 3A: T Cell Populations.
Fig. 3B: Myloid Cell Populations
Fig. 3B: Myloid Cell Populations.

Table 1:  Immune Cell Profiling of Naïve MC38 Tumors

Animal

CD45+
(%Total Cells)
CD4+T
(%CD3+)
CD8+T
(%CD3+)
Tregs
(%CD4+T)
Ratio
(CD8+ T/Tregs)
M1 MAC
(%CD11b+)
M2 MAC
(%CD11b+)
G-MDSC
(%CD11b+)
M-MDSC
(%CD11b+)
M1 3.94 41.4 44.1 12.8 8.33 29.7 3.06 3.18 12.5
M2 4.98 56.4 28.7 11.2 4.54 13.9 2.49 3.28 13.7
M3 14 36.4 50.9 23 6.07 11.7 15.3 11.5 21.3
M4 2.32 61.5 25.6 9.32 4.45 14.5 10.5 6.03 7.54
M5 1.48 52.1 30 19.7 2.93 14.3 15.8 18.7 10.6
Mean 5.3 49.6 35.9 15.2 5.3 16.8 9.4 8.5 13.1
SD 5 10.4 11 5.9 2.0 7.3 6.4 6.6 5.1

The MC38 murine colon carcinoma model has a favorable immune profile and can be employed as a robust preclinical immuno-oncology model. Our data supports the use of this tool in investigating novel treatment combinations with radiation, checkpoint inhibitors, or costimulatory molecules. Please contact MI Bioresearch to speak with our scientists about how MC38 or one of our other syngeneic models can be used for your next immuno-oncology study.

1Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2014, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2014/, based on November 2016 SEER data submission, posted to the SEER web site, April 2017.

2Corbett TH, Griswold Jr, DP, Roberts JC, Peckham JC, Schabel, Jr, FM (1975) Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen structure.  Cancer Research, 35: 2434-2439.

Sheri Barnes, PhDAbout the Author: Sheri Barnes joined MI Bioresearch in 2017 as Director, Scientific Development.  Prior to joining MI Bioresearch, she served as a Study Director at Charles River Laboratories. She has thirteen years of experience in the CRO industry, with the past ten years focused on using in vivo oncology research models in drug development. Sheri holds a Ph.D. in Cell and Developmental Biology from University of North Carolina at Chapel Hill.