Adoptive NK Cell Transfer as a Treatment in Colorectal Cancer Patients: Analyses of Tumour Cell Determinants Correlating With Efficacy In Vitro and In Vivo

Background Colorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC. Methods NK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity. Results HLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA− CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control. Conclusions Our results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy.

[1]  S. Gruber,et al.  Tumor immune infiltration estimated from gene expression profiles predicts colorectal cancer relapse , 2021, Oncoimmunology.

[2]  A. Poggi,et al.  Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis , 2020, Frontiers in Immunology.

[3]  C. Robert.  A decade of immune-checkpoint inhibitors in cancer therapy , 2020, Nature Communications.

[4]  N. Tumino,et al.  Glucocorticoids and the cytokines IL-12, IL-15 and IL-18 present in the tumor microenvironment induce PD-1 expression on human Natural Killer cells. , 2020, The Journal of allergy and clinical immunology.

[5]  R. Negrin,et al.  Indirect Impact of PD-1/PD-L1 Blockade on a Murine Model of NK Cell Exhaustion , 2020, Frontiers in Immunology.

[6]  J. Pardo,et al.  Recalling the Biological Significance of Immune Checkpoints on NK Cells: A Chance to Overcome LAG3, PD1, and CTLA4 Inhibitory Pathways by Adoptive NK Cell Transfer? , 2020, Frontiers in Immunology.

[7]  G. Lesinski,et al.  Immune checkpoint inhibitors for the treatment of MSI-H/MMR-D colorectal cancer and a perspective on resistance mechanisms , 2019, British Journal of Cancer.

[8]  R. Johnstone,et al.  Natural Killer Cells Suppress T Cell-Associated Tumor Immune Evasion. , 2019, Cell reports.

[9]  T. Waldmann,et al.  First-in-human trial of rhIL-15 and haploidentical natural killer cell therapy for advanced acute myeloid leukemia. , 2019, Blood advances.

[10]  B. A. Aksoy,et al.  Anti–CTLA-4 Activates Intratumoral NK Cells and Combined with IL15/IL15Rα Complexes Enhances Tumor Control , 2019, Cancer Immunology Research.

[11]  Marieke E. Ijsselsteijn,et al.  Revisiting immune escape in colorectal cancer in the era of immunotherapy , 2019, British Journal of Cancer.

[12]  D. Beebe,et al.  Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model , 2018, Oncoimmunology.

[13]  M. McBurney,et al.  Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade , 2018, The Journal of clinical investigation.

[14]  A. Copik,et al.  PD-L1 blockade enhances anti-tumor efficacy of NK cells , 2018, Oncoimmunology.

[15]  D. Jackson,et al.  MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma , 2018, Science Translational Medicine.

[16]  R. Weinberg,et al.  Understanding the tumor immune microenvironment (TIME) for effective therapy , 2018, Nature Medicine.

[17]  A. Prats,et al.  Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression , 2018, Oncoimmunology.

[18]  M. Berger,et al.  Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy , 2018, Science.

[19]  Wenfu Zhou,et al.  Predictive value of different proportion of lesion HLA-G expression in colorectal cancer , 2017, Oncotarget.

[20]  F. Marincola,et al.  Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications , 2017, International journal of molecular sciences.

[21]  L. Galluzzi,et al.  Control of Metastasis by NK Cells. , 2017, Cancer cell.

[22]  A. Jemal,et al.  Colorectal cancer statistics, 2017 , 2017, CA: a cancer journal for clinicians.

[23]  I. Melero,et al.  Targeting NK-cell checkpoints for cancer immunotherapy. , 2017, Current opinion in immunology.

[24]  A. Muntasell,et al.  Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells , 2016, Front. Immunol..

[25]  P. Laurent-Puig,et al.  Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression , 2016, Genome Biology.

[26]  D. Olive,et al.  PD-1 mediates functional exhaustion of activated NK cells in patients with Kaposi sarcoma , 2016, Oncotarget.

[27]  A. Varghese.  Chemotherapy for Stage II Colon Cancer , 2015, Clinics in Colon and Rectal Surgery.

[28]  Jeffrey S. Morris,et al.  The Consensus Molecular Subtypes of Colorectal Cancer , 2015, Nature Medicine.

[29]  Bert Vogelstein,et al.  PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. , 2015, The New England journal of medicine.

[30]  A. Björklund,et al.  Therapeutic Potential and Challenges of Natural Killer Cells in Treatment of Solid Tumors , 2015, Front. Immunol..

[31]  A. Muntasell,et al.  Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells , 2015, Oncoimmunology.

[32]  L. Terracciano,et al.  NK cells and T cells cooperate during the clinical course of colorectal cancer , 2014, Oncoimmunology.

[33]  F. Marincola,et al.  Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours , 2013, The Journal of pathology.

[34]  Justin Guinney,et al.  GSVA: gene set variation analysis for microarray and RNA-Seq data , 2013, BMC Bioinformatics.

[35]  M. Karamouzis,et al.  A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer , 2010, Cancer Immunology, Immunotherapy.

[36]  M. Ilyas,et al.  Intratumoral T cell infiltration, MHC class I and STAT1 as biomarkers of good prognosis in colorectal cancer , 2010, Gut.

[37]  Zhuang Liu,et al.  Drug delivery with carbon nanotubes for in vivo cancer treatment. , 2008, Cancer research.

[38]  M. Kloor,et al.  Immunoselective pressure and human leukocyte antigen class I antigen machinery defects in microsatellite unstable colorectal cancers. , 2005, Cancer research.

[39]  C. Le,et al.  Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. , 2005, Blood.

[40]  Hein Putter,et al.  Down-Regulation of HLA-A Expression Correlates with a Better Prognosis in Colorectal Cancer Patients , 2002, Laboratory Investigation.

[41]  R. Kerbel,et al.  Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. A. Sáez,et al.  The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma , 1997, Cancer.

[43]  S. Ogino,et al.  Molecular classification and correlates in colorectal cancer. , 2008, The Journal of molecular diagnostics : JMD.

[44]  F. Garrido,et al.  The HLA crossroad in tumor immunology. , 2000, Human immunology.

[45]  Andrew E. Jaffe,et al.  Bioinformatics Applications Note Gene Expression the Sva Package for Removing Batch Effects and Other Unwanted Variation in High-throughput Experiments , 2022 .