Chemotactic factors underlying tumor infiltration by immunocompetent cells in human colorectal cancer

Colorectal cancer (CRC) is a common digestive tract malignancy and a major cause of cancer
mortality. Several studies have convincingly shown that CRC infiltration by
immunocompetent cells and, in particular, cytotoxic CD8+ T cells (CTLs), IFN-γ-producing
T-helper 1 cells (Th1), Foxp3+ regulatory T cells (Tregs), and CD16+ MPO+ neutrophils, is
significantly associated with prolonged patient survival. However, the chemotactic factors
driving these cell populations into the tumor site, their cellular sources and their
microenvironmental triggers remain to be elucidated.
During my PhD training I have investigated the chemokine/chemokine receptor network
promoting CRC infiltration by immune cells associated to favorable prognosis.
In particular, I addressed:
1. The expression of immune cell markers and their correlation with chemokine
expression in primary CRC tissues;
2. The identification of chemokine receptors relevant for CRC infiltration by beneficial
immune cells;
3. The chemokine sources in CRC;
4. The microenvironmental stimuli triggering chemokine production in CRC tissues;
5. The effects of chemokine production on immune cell recruitment into CRC.
The expression of a panel of genes encoding 39 chemokines and 7 markers specific for
defined immune cell populations was assessed by quantitative PCR array in 62 samples of
freshly excised primary CRC and autologous healthy colonic tissue. Correlations between
expression of chemokine genes and immune cell markers were then evaluated.
Furthermore, chemokine receptor profiles were analysed by flow cytometry on cell
suspensions obtained upon digestion of clinical specimens or on corresponding cell
populations from autologous peripheral blood. Based on chemokine receptor expression on
tumor infiltrating cells and correlations between expression of chemokines and immune cell
markers, I could identify for each immune cell subset a putative “chemokine signature”:
1) CCL3, CCL5, CCL8 CXCL9, CXCL10 and CXCL12, associated with recruitment of
cytotoxic CTLs;
2) CCL5, CCL22, CXCL9, and CXCL12 correlating with infiltration by Th1;
3) CCL22 and CXCL12 potentially attracting Tregs;
4) CXCL2 and CXCL5 promoting chemotaxis of CD16+ MPO+ neutrophils.
I have further investigated potential chemokine sources and stimuli leading to chemokine
release within CRC tissues. I found that CRC cells purified from primary tumor specimens
express many of the genes encoding identified immune cell recruiting chemokines, including
CCL3, CCL5, CXCL2, CXCL5, CXCL9 and CXCL10. In vitro experiments showed that
chemokine production by CRC cells is triggered upon their exposure to microbial stimuli,
such as Toll-like receptor agonists, or CRC-associated bacteria, including Fusobacterium
nucleatum, Bacteroides Fragilis, Bacteroides vulgatus, and Escherichia Coli, thus suggesting
that components of the gut flora may critically influence chemokine production in CRC
tissues. This was indeed confirmed by “in vivo” experiments showing that chemokine gene
expression in xenografts, generated upon injection of human CRC cells in immunodeficient
NSG mice, appeared to be related to the presence of commensal bacteria. In particular,
chemokine gene expression levels in intracecal xenografts, were found to be ≥10 fold higher
as compared to those of subcutaneous xenografts, and they were significantly reduced upon
antibiotic treatment of tumor bearing mice.
Most importantly, a correlation between extent of immune cell infiltration and bacterial load
was also observed in human CRC samples. Indeed, CRC samples characterized by high
expression of chemokine and immune cell markers, displayed significantly higher bacterial
loads, as assessed by analysis of bacterial 16S ribosomal RNA, as compared to samples
showing low chemokine expression and immune cell infiltration. In addition, a significant
correlation between bacterial load and expression of the Th1 marker IRF1, CCL3 and CCL5,
was also detected.
Our in vitro and in vivo results cumulatively suggest that bacteria-induced chemokine
production by tumor cells may lead to tumor infiltration by beneficial immune cells.
Consistent with this hypothesis, in preliminary “in vitro” experiments, I found that
supernatants of bacteria-stimulated CRC cells promote chemotaxis of CTLs and Th1 cells to a
higher extent than untreated tumor cells.
Additional “in vivo” studies are clearly warranted. In particular, I plan to evaluate
intratumoral recruitment of CRC-derived CTLs and Th1 cells upon adoptively transfer into
intracecal xenografts-bearing mice.
Bacterial species or strains mostly contributing to high chemokine expression and immune
cell infiltration in human CRC samples also remain to be identified. Microbiome analysis of
CRC samples characterized by high or low immune cell infiltration might be envisaged in
future studies.
The results of the present work together with the proposed additional studies will contribute to
the understanding of the interplay occurring between gut flora and immune system in CRC,
and may pave the way towards innovative treatments aimed at modifying the gut flora in
order to promote CRC infiltration by beneficial immune cell subsets.