Regulatory T cells and cytotoxic T cells close to the epithelial-stromal interface are associated with a favorable prognosis

Julian Rudolf, Maike Büttner-Herold, Katharina Erlenbach-Wünsch, Rebecca Posselt, Jonas Jessberger, Marlen Haderlein, Markus Hecht, Arndt Hartmann, Rainer Fietkau, Luitpold Distel, Julian Rudolf, Maike Büttner-Herold, Katharina Erlenbach-Wünsch, Rebecca Posselt, Jonas Jessberger, Marlen Haderlein, Markus Hecht, Arndt Hartmann, Rainer Fietkau, Luitpold Distel

Abstract

Cytotoxic T cells and regulatory T cells play a crucial role in the outcome of cancer patients. Besides the density of these cells, it was shown recently that the spatial distribution is equally important. Here, we specifically analyzed the spatial distribution of these T cell subtypes at the epithelial-stromal interface in a rectal cancer cohort and its relevance for prognosis. We studied a cohort of 191 patients with advanced rectal cancer treated by radiochemotherapy (RCT). Tissue microarrays were immunohistochemical double-stained by FoxP3+ and CD+. Cell densities were analyzed in the stromal and epithelial compartment. Additionally, an image analysis software calculated the distances of lymphocytes to the epithelial-stromal interface (ESI). CD8+ and FoxP3+ cell counts decreased clearly after RCT with the decrease of FoxP3+ being more pronounced than of CD8+ cells. In the invasive front, short distances of the ESI to CD8+ and to FoxP3+ cells were associated with improved overall survival. Cell counts in the stromal compartment had no influence on prognosis. No correlation between stromal and epithelial lymphocyte densities was observed. The distance of epithelial-stromal interface to CD8+ and FoxP3+ cells was more accurate in predicting prognosis in the stromal compartment of rectal cancer patients than mere cell counts and could thereby be means of better stratifying patients for therapy. This observation will have to be validated in future prospective studies with regard to other tumor entities and its implications for the responsiveness of tumors to new therapeutic modalities.

Keywords: epithelial–stromal interface; rectal cancer; regulatory T cells; spatial distribution; cytotoxic T cells.

© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.

Figures

Figure 1.
Figure 1.
Clinical characteristics of the cohort and TMA samples: (a) Kaplan Meier plots of overall survival, recurrence-free survival and metastasis-free survival in the cohort. (b) Distant metastasis and overall survival. (c) Regression grade (Dworak) and overall survival. Tissue samples were processed into microarrays with a core diameter of 2 mm. (d) Here, an example of tissue derived from the center of a tumor is depicted. (e) The epithelial compartment of the tumor was marked manually using Biomas software. (f) Examples of CD8+ and FoxP3+ T-cells; TIL in proximity to the ESI. (g) Density of stromal TILs in different compartments. (h) Distances of ESI to TIL in different stromal compartments.
Figure 2.
Figure 2.
Kaplan-Meier-Plots for overall survival depending on the stromal TIL density and the stromal-located lymphocyte distances to the epithelial–stromal interface (ESI) in different compartments: (a) Density of CD8+ TIL in the biopsy. (b) Density of CD8+ TIL in the invasive front. (c) Density of CD8+ TIL in the central tumor area. (d) ESI to CD8+ distances in the biopsy. (e) ESI to CD8+ distances in the invasive front. (f) ESI to CD8+ distances in the central tumor area. (g) Density of FoxP3+ TIL in the biopsy. (h) Density of FoxP3+ TIL in the invasive front. (j) Density of FoxP3+ TIL in the central tumor area. (k) ESI to FoxP3+ distances in the biopsy. (l) ESI to FoxP3+ distances in the invasive front. (m) ESI to FoxP3+ distances in the central tumor area.
Figure 3.
Figure 3.
Distances of ESI to TIL depending on their density in different tumor compartments: (a) Density of FoxP3+ TIL in the stroma and ESI to FoxP3+ TIL distance. (b) Density of intraepithelial FoxP3+ TIL and ESI to FoxP3+ TIL distance. (c) Density of CD8+ TIL in the stroma and ES to CD8+ TIL distance. (d) Density of intraepithelial CD8+ TIL and ESI to CD8+ TIL distance. (e) Intraepithelial vs stromal FoxP3+ density. (f) Intraepithelial vs stromal CD8+ density.
Figure 4.
Figure 4.
Cell to cell distances and cell densities in the ESI to CD8+ and ESI to FoxP3+ samples of the invasive front: Cell to cell distances and cell densities in the samples with ESI to CD8+

Figure 5.

Changes in distances of ESI…

Figure 5.

Changes in distances of ESI to TIL comparing biopsies and tumor resections: (a)…

Figure 5.
Changes in distances of ESI to TIL comparing biopsies and tumor resections: (a) ESI to TIL distance changes depending on the compartment [µm]. (b) Changes of ESI to TIL distance depending on the compartment [number of patients]. (c) Change of ESI to FoxP3+ TIL distance in the invasive front regarding overall survival. (d) Change of ESI to CD8+ TIL distance in the invasive front regarding overall survival. (e) Change of ESI to FoxP3+ TIL distance in the central tumor area regarding overall survival. (f) Change of ESI to CD8+ TIL distance in the central tumor area regarding overall survival.
Figure 5.
Figure 5.
Changes in distances of ESI to TIL comparing biopsies and tumor resections: (a) ESI to TIL distance changes depending on the compartment [µm]. (b) Changes of ESI to TIL distance depending on the compartment [number of patients]. (c) Change of ESI to FoxP3+ TIL distance in the invasive front regarding overall survival. (d) Change of ESI to CD8+ TIL distance in the invasive front regarding overall survival. (e) Change of ESI to FoxP3+ TIL distance in the central tumor area regarding overall survival. (f) Change of ESI to CD8+ TIL distance in the central tumor area regarding overall survival.

References

    1. Nakano O, Sato M, Naito Y, Suzuki K, Orikasa S, Aizawa M, Suzuki Y, Shintaku I, Nagura H, Ohtani H.. Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity. Cancer Res. 2001;61:5132–9.
    1. Sharma P, Shen Y, Wen S, Yamada S, Jungbluth AA, Gnjatic S, Bajorin DF, Reuter VE, Herr H, Old LJ, et al. CD8 tumor-infiltrating lymphocytes are predictive of survival in muscle-invasive urothelial carcinoma. Proc Natl Acad Sci U S A. 2007;104(10):3967–3972. doi:10.1073/pnas.0611618104.
    1. Posselt R, Erlenbach-Wunsch K, Haas M, Jessberger J, Buttner-Herold M, Haderlein M, Hecht M, Hartmann A, Fietkau R, Distel LV. Spatial distribution of FoxP3+ and CD8+ tumour infiltrating T cells reflects their functional activity. Oncotarget. 2016;7(37):60383–60394. doi:10.18632/oncotarget.11039.
    1. Chew A, Salama P, Robbshaw A, Klopcic B, Zeps N, Platell C, Lawrance IC. SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer. PLoS One. 2011;6(7):e22047. doi:10.1371/journal.pone.0022047.
    1. Correale P, Rotundo MS, Del Vecchio MT, Remondo C, Migali C, Ginanneschi C, Tsang KY, Licchetta A, Mannucci S, Loiacono L, et al. Regulatory (FoxP3+) T-cell tumor infiltration is a favorable prognostic factor in advanced colon cancer patients undergoing chemo or chemoimmunotherapy. J Immunother. 2010;33(4):435–441. doi:10.1097/CJI.0b013e3181d32f01.
    1. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol. 2009;27(2):186–192. doi:10.1200/JCO.2008.18.7229.
    1. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol. 2010;10(7):490–500. doi:10.1038/nri2785.
    1. Carstens JL, Correa de Sampaio P, Yang D, Barua S, Wang H, Rao A, Allison JP, LeBleu VS, Kalluri R. Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer. Nat Commun. 2017;8:15095. doi:10.1038/ncomms15095.
    1. Berthel A, Zoernig I, Valous NA, Kahlert C, Klupp F, Ulrich A, Weitz J, Jaeger D, Halama N. Detailed resolution analysis reveals spatial T cell heterogeneity in the invasive margin of colorectal cancer liver metastases associated with improved survival. Oncoimmunology. 2017;6(3):e1286436. doi:10.1080/2162402X.2017.1286436.
    1. Jager T, Neureiter D, Fallaha M, Schredl P, Kiesslich T, Urbas R, Klieser E, Holzinger J, Sedlmayer F, Emmanuel K, et al. The potential predictive value of tumor budding for neoadjuvant chemoradiotherapy response in locally advanced rectal cancer. Strahlenther Onkol. 2018;194(11):991–1006. doi:10.1007/s00066-018-1340-0.
    1. Paleny R, Bremer M, Walacides D, Mainwaring S, Weber K, Henkenberens C. Comparison of relative and absolute rectal dose-volume parameters and clinical correlation with acute and late radiation proctitis in prostate cancer patients. Strahlenther Onkol. 2019;195(2):103–112. doi:10.1007/s00066-018-1365-4.
    1. Feichtenbeiner A, Haas M, Buttner M, Grabenbauer GG, Fietkau R, Distel LV. Critical role of spatial interaction between CD8(+) and Foxp3(+) cells in human gastric cancer: the distance matters. Cancer Immunol Immunother. 2014;63(2):111–119. doi:10.1007/s00262-013-1491-x.
    1. Nagl S, Haas M, Lahmer G, Büttner-Herold M, Grabenbauer GG, Fietkau R, Distel L. Cell-to-cell distances between tumour infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells. Oncoimmunology. 2016;5(5):e1127494. doi:10.1080/2162402X.2015.1127494.
    1. Heindl A, Nawaz S, Yuan Y. Mapping spatial heterogeneity in the tumor microenvironment: a new era for digital pathology. Lab Invest. 2015;95(4):377–384. doi:10.1038/labinvest.2014.155.
    1. Echarti A, Hecht M, Buttner-Herold M, Haderlein M, Hartmann A, Fietkau R, Distel L. CD8+ and regulatory T cells differentiate tumor immune phenotypes and predict survival in locally advanced head and neck cancer. Cancers (Basel). 2019;11(9):1398. doi:10.3390/cancers11091398.
    1. Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541(7637):321-330. doi: 10.1038/nature21349.

Source: PubMed

Sponsors et collaborateurs

Les conditions médicales

Interventions en matière de drogue

3
S'abonner