TissueFAXS Chroma and StrataQuest analysis to identify potential targets for therapies against colorectal liver metastases

Colorectal cancer is the second most common cause of cancer-related mortality.¹ A significant contributing factor to this statistic is the high risk of liver metastases, which can affect up to half of patients with colorectal cancer as treatment options are limited.² The mainstay of treatment for patients with colorectal liver metastases (CRLMs) is liver surgery, but this is not feasible for all patients. Alternative treatment options are thus being sought.

One novel approach in cancer treatment is to target the anti or pro tumoral immune response. Tumor infliltrating immune cells include cellular populations with a wide range of phenotypes and functions.³ For example, the profile and distribution of intra-tumoral T cells (TILs) are key parameters for evaluating prognosis in primary colorectal cancer.⁴ Furthermore, evidence indicates that a favorable prognosis in CRLM correlates with CD8+TIL levels in CRLM.^5,6 In terms of clinical prognosis, also other immune cell types such as B cells play an important role since they form B-cell-enriched and highly proliferative immune structures in the vicinity of cancer tissue, which are shown to be prognostic towards an improved clinical outcome. ⁷

In respect to T cells, a high concentration of intra-tumoral CD8+ tissue-resident memory T cells (TRM cells) has been linked to improved survival in primary colorectal cancer.⁸

As more studies indicate the importance of  TILs in anti-tumor responses, they may be a potential target for novel treatment approaches in CRLM. To facilitate the development of such alternative therapeutic options, the characterization of tumor-reactive CD8+ TILs was required. In particular, it would be helpful for the determination of significant differences in TIL composition between healthy liver and CRLM.

It has been reported that CD103+CD39+CD8+ TRM cells are present at higher levels in CRLM relative to adjacent liver tissue.⁹ These specific subpopulations of CD8 cells have now been evaluated in detail using the TissueFAXS CHROMA imaging platform¹⁰ and StrataQuest image analysis software.¹¹

Evaluating CD8 cells in colorectal liver metastases

A recent study using TissueFAXS CHROMA and StrataQuest evaluated the frequencies and activation states of CD103+CD8+ TRM cells and other T cell populations in CRLM and adjacent liver tissue in samples from patients with CRC. Further it investigated whether CD8+ TRM cells accumulated in subcutaneous CRLM-derived tumors in lymphocyte-deficient mice¹².

The samples were imaged as entire tissue at 20x magnification using the automated scanning function of TissueFAXS CHROMA¹⁰.

The images obtained by TissueFAXS CHROMA were analyzed using the StrataQuest software.¹¹DAPI staining was used to detect individual cells, and specifc marker staining were used to identify specific cell phenotypes. Following a machine-learning process, the StrataQuest software made it possible to separate tumor cells from stromal cells. Furthermore, StrataQuest enabled to group stromal cells according to their distance from the tumor.

Findings

The TissueFAXS CHROMA and StrataQuest technologies facilitated the identification of distinct immune microenvironments of CRLM and the adjacent liver. CRLM tissue samples showed increased frequencies of CD4+ αβ T cells and decreased levels of CD8+ αβ T cells.¹²

Investigation of the capacity of the predominant TILs to control the growth of CRLM-derived tumors in vivo using a patient-derived xenograft model revealed that the transferred TILs only achieved control of CRLM-derived tumors after trametinib treatment. Interestingly, rather than accumulating in the blood or spleen, CD103+CD8+ TRM cells strictly accumulated within the autologous CRLM-derived tumor.

The prevalence of CD103+CD39+CD8+ TRM cells in CRLM in relation to the adjacent liver and the susceptibility of CD103+CD8+ TRM cells for the repopulation of the autologous tumor provides evidence to suggest that further investigation into the viability of CD103+CD39+CD8+ TRM as potential targets for treatments against advanced CRLM is needed.

Summary

CD103+CD39+CD8+ TRM cells accumulated in CRLM relative to adjacent liver tissue, highlighting them as potential targets for the design of novel treatment approaches for CRLM.

These phenotypic and functional analyses of TILs in CRLM using TissueFAXS CHROMA and StrataQuest may serve to inform the development of novel TIL-mediated treatments for colorectal cancer and liver metastases.

This new data obtained using TissueFAXS CHROMA and StrataQuest is of particular importance as it highlights their potential to assist in research addressing the treatment of advanced colorectal cancer in patients with inoperable liver metastases.

TissueFAXS CHROMA – multipectal high-speed imaging

TissueFAXS CHROMA is a versatile multifluorescence imaging platform that simultaneously scans the whole slides for up to 7 markers without bleed through. TissueFAXS CHROMA comes with the high-end image analysis solution StrataQuest and thereby provides a holistic solution for cellular phenotyping and in-depth tissue microenvironment characterization.

StrataQuest

The StrataQuest software¹¹ provides a multitude of streamlined analysis solution including the sophisticated cell-to-cell contact App. The App allows to identify the cellular phenotype of specific fluorescence-stained cell populations and establish the cellular contacts to their neighboring cells. It readily provides the staining intensity per marker and morphometric parameters for each segmented cell/cell compartment. Furthermore, as was important in this research, StrataQuest makes it possible to detect cells in situ that are defined by more than one marker, in this case, CD3+, CD103+, CD8+, and TRM.

The StrataQuest analysis is conducted using a Cell to Cell Contact App available online in the TissueGnostics App Center.

References and Further Reading

1. Sung H, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249.
2. Martin J, et al. Colorectal liver metastases: Current management and future perspectives. World J. Clin. Oncol. 2020, 11, 761–808.
3. Bartsch LM, et al. Tissue-Resident Memory T Cells in the Liver-Unique Characteristics of Local Specialists. Cells 2020, 9, 2457.
4. Pages F, et al. In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J. Clin. Oncol. 2009,27, 5944–5951.
5. Liang JY, et al. Histopathological growth patterns correlate with the immunoscore in colorectal cancer liver metastasis patients after hepatectomy. Cancer Immunol. Immunother. 2020, 69, 2623–2634.
6. Wang E, et al. Prognostic value of the density of tumor-infiltrating lymphocytes in colorectal cancer liver metastases. Oncol. Lett. 2021, 22, 837.
7. Mungenast, F. et al. The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer. Cancers 2020, 12, 3117. https://doi.org/10.3390/cancers12113117
8. Okla K, et al, Tissue-resident memory T cells in tumor immunity and immunotherapy. J. Exp. Med. 2021,218, e20201605.
9. Liang F, et al. Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells. Cancers 2021, 13, 5247.
10. https://tissuegnostics.com/products/multispectral-cytometer/tissuefaxs-chroma
11. https://tissuegnostics.com/products/contextual-image-analysis/strataquest-apps
12. Liang F, et al. A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells. Cancers 2022, 14,2882.

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