An Investigation to Study Morphology, Nanomechanical Attributes, Transcriptomics, and Proteomics of Tumor Associated Macrophages Derived Extracellular Vesicles in the Tumor Microenvironment

• T.K. Kulkarni

  Mayo Clinic, Jacksonville, FL, USA

• A. Banerjee

  Boston Medical School, Boston, MA, USA

• N. Banerjee

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• J. Cuffee

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• S. Newell

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• E. Armstrong

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• S.N. Deloatch

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• J. Jong Park

  Moffit Cancer Center, Tampa, FL, USA

• A.H. El-Hashash

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

• S. Bhattacharya

  Mayo Clinic, Jacksonville, FL, USA

• H. Banerjee

  Elizabeth City State University Campus of The University of North Carolina, Elizabeth City, NC, USA

Introduction: This study investigates the molecular and biophysical changes in Tumor-Associated Macrophage (TAM)-derived exosomes (TAME) compared to exosomes from non-tumor microenvironment (TE) macrophages (MO-E). TAMs are immune cells infiltrating tumors that promote cancer progression.

Materials and Methods: Characterizing the cargo of TAME via Next Generation RNA Sequencing protein microarray analysis and studying their morphological and nanomechanical properties using Atomic Force Microscopy (AFM).

Results: Key Findings regarding Morphology showed TAME are significantly smaller (~50.6 nm) than MO-E (~64.8 nm).

TAME exhibit higher Young's modulus (~15.5 MPa) indicating increased stiffness. Surface roughness ot TAME Is slightly higher (~3.64 nm) than MO-E (~3.51 nm). RNA sequencing revealed differential expression of genes involved in drug metabolism, cell cycle regulation, survival pathways, and immune modulation. Pathways such as NF-KB, PI3K/Akt, IL-13 signaling, and metabolic reprogramming are influenced by TAME cargo.

Proteomics studies showed several Top upregulated proteins include ORP150 (promotes VEGF secretion), CSRP1, C1qB, DNER, PEPD, with roles in tumor growth and metastasis.

Downregulated proteins include tumor suppressors like GOLPH2 and VNN1.

These protein profiles suggest TAME carry oncoproteins that support tumor proliferation, invasion, and immune evasion.

Conclusion: This study demonstrates that TAM-derived exosomes are morphologically distinct and carry molecular cargo that modulates key cancer-related pathways, highlighting their potential as biomarkers and therapeutic targets. The significance lies in understanding TAME's role in cancer progression, which could lead to novel diagnostic biomarkers and therapeutic targets, especially considering current limitations in cancer diagnosis accuracy.

[1] Wang J, Li D, Cang H, Guo B. Crosstalk between cancer and immune cells: Role of tumor-associated macrophages in the tumor microenvironment. Cancer Med 2019; 8(10): 4709-4721.

[2] Caronni N, La Terza F, Vittoria FM, et al. IL-1β+ macrophages fuel pathogenic inflammation in pancreatic cancer. Nature 2023; 623(7986): 415-422.

[3] Banerjee H, Krauss C, Worthington M, Banerjee N, Walker RS, Hodges S, Chen L, Rawat K, Dasgupta S, Ghosh S, Mandal S. Differential expression of efferocytosis and phagocytosis associated genes in tumor associated macrophages exposed to African American patient derived prostate cancer microenvironment. J Solid Tumors 2019; 9(2): 22-27.

[4] Xiang X, Wang J, Lu D, et al. Targeting tumor-associated macrophages to synergize tumor immunotherapy. Signal Transduct Target Ther 2021; 6(1): 75.

[5] Zhang S, Fang W, Zhou S. Single cell transcriptomic analyses implicate an immune-suppressive tumor microenvironment in pancreatic cancer liver metastasis. Nat Commun 2023; 14(1): 5123.

[6] Kemp SB, Steele NG, Carpenter ES, et al. Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages. Life Sci Alliance 2021; 4(6): e202000935.

[7] Yu M, Guan R, Hong W, et al. Prognostic value of tumor-associated macrophages in pancreatic cancer: a meta-analysis. Cancer Manag Res 2019; 11: 4041-4058.

[8] He Z, Wang J, Zhu C, et al. Exosome-derived FGD5-AS1 promotes tumor-associated macrophage M2 polarization-mediated pancreatic cancer cell proliferation and metastasis. Cancer Lett 2022; 548: 215751.

[9] Zhu Y, Knolhoff BL, Meyer MA, et al. CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models. Cancer Res 2014; 74(18): 5057-69.

[10] Halbrook CJ, Pontious C, Kovalenko I, et al. Macrophage-released pyrimidines inhibit gemcitabine therapy in pancreatic cancer. Cell Metab 2019; 29(6): 1390-1399.e6.

[11] Alonso-Nocelo M, Ruiz-Cañas L, Sancho P, et al. Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma. Gut 2023; 72(2): 345-359.

[12] Zhang M, Pan X, Fujiwara K, et al. Pancreatic cancer cells render tumor-associated macrophages metabolically reprogrammed by a GARP and DNA methylation-mediated mechanism. Signal Transduct Target Ther 2021; 6(1): 366.

[13] Wang W, Marinis JM, Beal AM, et al. RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer. Cancer Cell 2018; 34(5): 757-774.e7.

[14] Farajzadeh VS, Keshavarz-Fathi M, Silvestris N, et al. The role of inflammatory cytokines and tumor associated macrophages (TAMs) in microenvironment of pancreatic cancer. Cytokine Growth Factor Rev 2018; 39: 46-61.

[15] Parayath NN, Hong BV, Mackenzie GG, et al. Hyaluronic acid nanoparticle-encapsulated microRNA-125b repolarizes tumor-associated macrophages in pancreatic cancer. Nanomedicine (Lond) 2021; 16(25): 2291-2303.

[16] Cui R, Yue W, Lattime EC, et al. Targeting tumor-associated macrophages to combat pancreatic cancer. Oncotarget 2016; 7(31): 50735-50754.

[17] Gu H, Deng W, Zhang Y, et al. NLRP3 activation in tumor-associated macrophages enhances lung metastasis of pancreatic ductal adenocarcinoma. Transl Lung Cancer Res 2022; 11(5): 858-868.

[18] Ho TT, Nasti A, Seki A, et al. Combination of gemcitabine and anti-PD-1 antibody enhances the anticancer effect of M1 macrophages and the Th1 response in a murine model of pancreatic cancer liver metastasis. J Immunother Cancer 2020; 8(2): e001367.

[19] Zhang K, Qin YH, Shen J, et al. [Progressin tumor-associated macrophages in the treatment of pancreatic cancer]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2023; 45(3): 471-478. Chinese.

[20] Kashfi K, Kannikal J, Nath N. Macrophage reprogramming and cancer therapeutics: Role of iNOS-derived NO. Cells 2021; 10(11): 3194.

[21] Shlyakhtenko LS, Gall AA, Lyubchenko YL. Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy. Cell Imaging Techniques: Springer 2012; pp. 295-312.

[22] Woo S, Rothemund PW. Self-assembly of two-dimensional DNA origami lattices using cation-controlled surface diffusion. Nature Commun 2014; 5(1): 1-11.

[23] Engler AJ, Rehfeldt F, Sen S, et al. Microtissue elasticity: measurements by atomic force microscopy and its influence on cell differentiation. Meth Cell Biol 2007; 83: 521-45.

[24] Jorba I, Uriarte JJ, Campillo N, et al. Probing micromechanical properties of the extracellular matrix of soft tissues by atomic force microscopy. J Cell Physiol 2017; 232(1): 19-26.

[25] Kulkarni T, Tam A, Mukhopadhyay D, et al. AFM study: Cell cycle and probe geometry influences nanomechanical characterization of Panc1 cells. Biochimica et Biophysica Acta (BBA)-General Subjects 2019; 1863(5): 802-12.

[26] Kulkarni T, Mukhopadhyay D, Bhattacharya S. Dynamic alteration of poroelastic attributes as determinant membrane nanorheology for endocytosis of organ specific targeted gold nanoparticles. J Nanobiotech 2022 20(1): 1-16.

[27] Kulkarni T, Angom RS, Das P. Nanomechanical insights: Amyloid beta oligomer-induced senescent brain endothelial cells. Biochimica et Biophysica Acta (BBA)-Biomembranes 2019; 1861(12): 183061.

[28] Kulkarni T, Mukhopadhyay D, Bhattacharya S. Nanomechanical Insight of Pancreatic Cancer Cell Membrane during Receptor Mediated Endocytosis of Targeted Gold Nanoparticles. ACS Applied Bio Materials 2020; 4(1): 984-94.

[29] Efremov YM, Okajima T, Raman A. Measuring viscoelasticity of soft biological samples using atomic force microscopy. Soft Matter 2020; 16(1): 64-81.

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