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Secretome profiling of heterotypic spheroids suggests a role of fibroblasts in HIF-1 pathway modulation and colorectal cancer photodynamic resistance

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Abstract

Purpose

Previous analyses of the tumor microenvironment (TME) have resulted in a concept that tumor progression may depend on interactions between cancer cells and its surrounding stroma. An important aspect of these interactions is the ability of cancer cells to modulate stroma behavior, and vice versa, through the action of a variety of soluble mediators. Here, we aimed to identify soluble factors present in the TME of colorectal cancer cells that may affect relevant pathways through secretome profiling.

Methods

To partially recapitulate the TME and its architecture, we co-cultured colorectal cancer cells (SW480, TC) with stromal fibroblasts (MRC-5, F) as 3D-spheroids. Subsequent characterization of both homotypic (TC) and heterotypic (TC + F) spheroid secretomes was performed using label-free liquid chromatography-mass spectrometry (LC-MS).

Results

Through bioinformatic analysis using the NCI-Pathway Interaction Database (NCI-PID) we found that the HIF-1 signaling pathway was most highly enriched among the proteins whose secretion was enhanced in the heterotypic spheroids. Previously, we found that HIF-1 may be associated with resistance of colorectal cancer cells to photodynamic therapy (PDT), an antitumor therapy that combines photosensitizing agents, O2 and light to create a harmful photochemical reaction. Here, we found that the presence of fibroblasts considerably diminished the sensitivity of colorectal cancer cells to photodynamic activity. Although the biological significance of the HIF-1 pathway of secretomes was decreased after photosensitization, this decrease was partially reversed in heterotypic 3D-spheroids. HIF-1 pathway modulation by both PDT and stromal fibroblasts was confirmed through expression assessment of the HIF-target VEGF, as well as through HIF transcriptional activity assessment.

Conclusion

Collectively, our results delineate a potential mechanism by which stromal fibroblasts may enhance colorectal cancer cell survival and photodynamic treatment resistance via HIF-1 pathway modulation.

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Abbreviations

3D:

Three dimensional

AGC:

Automatic gain control

CRC:

Colorectal cancer

DMSO:

Dimethyl sulfoxide

ENO1:

Enolase 1

F:

Fibroblast

FBS:

Fetal bovine serum

GFP:

Green fluorescent protein

GLUT-1:

Glucose transporter 1

HCD:

High energy collision induced dissociation

HIF-1:

Hypoxia inducible factor-1

LDHA:

Lactate dehydrogenase

LC-MS:

Liquid chromatography–Mass spectrometry

LFQ:

Label-free quantification

Me-ALA:

Aminolevulinic acid methyl ester

MTT:

1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan

NCI-PID:

National Cancer Institute–Pathway Interaction Database

NPM1:

Nucleophosmin

PAI-1:

Plasminogen activator inhibitor 1

PBS:

Phosphate buffer saline

PpIX:

Protoporphyrin IX

PS:

Photosensitizer

TC:

Tumor cell

TFRC:

Transferrin receptor protein 1

VEGF:

Vascular endothelial growth factor

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Acknowledgements

This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (PICT), Secretaría de Ciencia y Técnica (SECyT), Universidad Nacional de Río Cuarto, and a Christian Boulin Fellowship (EMBL). VAR and NBRV are members of the Scientific Researcher Career at CONICET. MJL holds a fellowship from CONICET.

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Authors and Affiliations

  1. Departamento de Biología Molecular, Facultad de Ciencias Exactas Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, 5800, Río Cuarto, Córdoba, Argentina

    María Julia Lamberti, Viviana Alicia Rivarola & Natalia Belén Rumie Vittar

  2. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Mandy Rettel & Jeroen Krijgsveld

  3. German Cancer Research Center (DKFZ), Heidelberg, Germany

    Jeroen Krijgsveld

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  1. María Julia Lamberti
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Correspondence to Viviana Alicia Rivarola or Natalia Belén Rumie Vittar.

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Table S1

Perseus correlation matrix. Correlation coefficients whose magnitude were greater than 0.7 were considered highly correlated (red numbers indicate correlation values <0.7) (DOCX 19 kb)

Table S2

List of proteins identified in homotypic spheroids secretome. Available information: ID identified according to UniProt, name of the protein, corresponding gene, location of the signal peptide in those proteins secreted by the classical pathway (SignalP), NN-score of proteins secreted by non-classical pathways (SecretomeP, the NN-score should be greater than 0.5), LFQ (“label-free quantification”) value (mean and standard deviation). (DOCX 33 kb)

Table S3

List of proteins identified in heterotypic spheroids secretome. Available information: ID identified according to UniProt, name of the protein, corresponding gene, location of the signal peptide in those proteins secreted by the classical pathway (SignalP), NN-score of proteins secreted by non-classical pathways (SecretomeP, the NN-score should be greater than 0.5), LFQ (“label-free quantification”) value (mean and standard deviation). (DOCX 36 kb)

Table S4

List of proteins identified in PDT-treated homotypic spheroids secretome. Available information: ID identified according to UniProt, name of the protein, corresponding gene, location of the signal peptide in those proteins secreted by the classical pathway (SignalP), NN-score of proteins secreted by non-classical pathways (SecretomeP, the NN-score should be greater than 0.5), LFQ (“label-free quantification”) value (mean and standard deviation). (DOCX 45 kb)

Table S5

List of proteins identified in PDT-treated heterotypic spheroids secretome. Available information: ID identified according to UniProt, name of the protein, corresponding gene, location of the signal peptide in those proteins secreted by the classical pathway (SignalP), NN-score of proteins secreted by non-classical pathways (SecretomeP, the NN-score should be greater than 0.5), LFQ (“label-free quantification”) value (mean and standard deviation). (DOCX 55 kb)

Fig S1

Histograms. Histograms were performed using the logarithmic LFQ values (log10 (x)) to visualize the normal distribution of the data (Perseus software) (JPG 63 kb)

Fig S2

Multiscatter plot. The multiscatter plot was performed using the logarithmic LFQ values (log2 (x)) to visualize the correlation between the replicates (JPG 220 kb)

Fig S3

Gene Ontology. (A) Biological process classification in Gene Ontology analysis of proteins whose secretion was enhanced in in photosensitized homotypic spheroids compared to untreated ones. (B) Biological process classification in Gene Ontology analysis of proteins whose secretion was enhanced in in photosensitized heterotypic spheroids compared to untreated ones. (PNG 980 kb)

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Lamberti, M.J., Rettel, M., Krijgsveld, J. et al. Secretome profiling of heterotypic spheroids suggests a role of fibroblasts in HIF-1 pathway modulation and colorectal cancer photodynamic resistance. Cell Oncol. 42, 173–196 (2019). https://doi.org/10.1007/s13402-018-00418-8

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  • DOI: https://doi.org/10.1007/s13402-018-00418-8

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