Background
Colorectal cancer (CRC) incidence and mortality have been decreasing in those who are 50 and older (late onset) over the last 30 years, however, the incidence of CRC in people less than 50 years old (early-onset) has increased from 8.6 to 12.9 per 100,00 from 1992 to 2018. Early onset CRC (EO-CRC) is hypothesized to have a distinct etiology from late-onset CRC (LO-CRC), yet both EO- and LO-CRC exhibit similar genetic mutations. As a result, researchers have started exploring the tumor microenvironment (TME) to explain the rise of EO-CRC. Immune cell co-culture of patient-derived organoids (PDO) with matched patient peripheral blood mononuclear cells (PBMCs) is a new model for evaluating the functional relationship between tumor and the TME. We aim to utilize this co-culture technique to tease out the unique inflammatory phenotype of EO-CRC.
Methods
We are expanding a biobank of CRC and adjacent normal tissue and PBMCs from patients with EO- and LO- CRC. We are generating PDO from these tissues for use in co-culture experiments. Our first aim is to demonstrate the ability to recapitulate the tumor microenvironment in vitro using PDO and patient-matched PBMCs. On day 1 of co-culture, PBMCs are flow sorted for macrophages (CD11b) and T cells (CD3, CD4, CD8). Cells are co-cultured with CRC patient-matched PDO at a 4:1 effector:target cell ratio. On day 5 of culture, co-cultured wells are collected for flow cytometry analysis.
Results
We have collected tumor and adjacent normal tissue samples from 29 patients (17% early onset). We have foundation testing on 26 samples and have identified 6 MSI-H tumors, 2 tumors with NRAS mutations, 6 with KRAS mutations, 3 with BRAF mutations, and 4 with PIK3CA mutations. We have generated 19 PDO lines thus far, with an 86% success rate (19/22).

On day of co-culture, PBMCs were flow sorted, demonstrating 97% viability. Of viable cells, 11.53% were macrophages (CD11b+) and 28.56% T cells (CD3+). The T cell population consisted of 67.02% CD4+ and 14.87% CD8+ (Fig 1).

On day 5 of co-culture, flow cytometry analysis demonstrated 94.85% viability. Of immune cells (EP-CAM negative), 54.87% were macrophages (CD11b+) and 32.99% were CD3+. Of CD3+ cells, 11.97% were CD4+, and 11.4% were CD8+. Overall, these findings demonstrate PBMC co-culture with CRC PDO retain major immune subsets (Fig 2).
Discussion
In our first in vitro experiments utilizing co-culture of CRC PDO with patient-matched PBMC, we demonstrated T cells and macrophages are retained in co-culture for up to 5 days. Our ability to recapitulate the tumor microenvironment of CRC in vitro opens the door for future experiments aimed at identifying differences between the immune phenotype of EO-CRC and LO-CRC. We will utilize this platform to exploit unique dependencies between the TME and tumor to develop and evaluate novel therapeutics and detection strategies.

Figure 1: <i>Flow Cytometry Analysis of PBMC</i><br /> Flow cytometry data from PBMC sort prior to co-culture experiment. PI was used to assess viability (B). CD11b was used as marker for macrophages (C, box D), while CD3 (C, box E), CD4 (E, box G), and CD8 (E, box F) were used as T cell markers.

Figure 1: Flow Cytometry Analysis of PBMC
Flow cytometry data from PBMC sort prior to co-culture experiment. PI was used to assess viability (B). CD11b was used as marker for macrophages (C, box D), while CD3 (C, box E), CD4 (E, box G), and CD8 (E, box F) were used as T cell markers.

<i>Figure 2: Flow Cytometry Analysis of Co-Culture</i><br /> On day 5 of co-culture, wells were collected and underwent flow cytometry analysis. PI was used to assess viability (B). EP-CAM was used as a marker for epithelial cells (C, box J). CD11b was used as a marker for macrophages (K, box M). CD3 (K, box L), CD4 (L, Q4), and CD8 (L, Q1) were used as T cell markers.

Figure 2: Flow Cytometry Analysis of Co-Culture
On day 5 of co-culture, wells were collected and underwent flow cytometry analysis. PI was used to assess viability (B). EP-CAM was used as a marker for epithelial cells (C, box J). CD11b was used as a marker for macrophages (K, box M). CD3 (K, box L), CD4 (L, Q4), and CD8 (L, Q1) were used as T cell markers.

Yosuke Mitani

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