Bone marrow-derived cells in angiogenesis and cancer

Tumor is more than a mass of transformed cells. Tumor cells are embedded in the supporting tumor stroma, that is composed of cellular and non-cellular components. The cellular components include vascular cells, bone marrow-derived cells (BMDCs), and fibroblasts. In concert, tumor cells and stromal cells secrete proteases, cytokines, chemokines, and growth factors that modulate the tumor behavior. The mechanisms how stromal cells contribute to the tumor growth and tumor angiogenesis are still incompletely understood. This thesis aimed to study the role of BMDCs in angiogenesis and cancer. First, the role of BMDCs as a source of vascular endothelium was examined. The second study addressed the effects of an EGFR inhibitor, gefitinib, on the vascular cells and BMDCs in an EGFR-deficient B16 mouse melanoma tumor model. In the third study, chemokine-like prokineticins, that may have both angiogenic and immunomodulatory properties, were examined in a virus-associated human skin cancer type, Merkel cell carcinoma (MCC). BMDCs did not incorporate into the vascular endothelium in any of the experimental models, suggesting that the pre-existing vasculature is the main source of endothelium during angiogenesis. The potential of the BMDCs to differentiate into vascular endothelium may require specific molecular microenvironments, which needs further analysis. Treatment of B16 tumors with the EGFR inhibitor gefitinib reduced the pericyte number and coverage in the small CD31+ capillaries and the numbers of perivascular BMDCs suggesting that gefitinib treatment might have vascular and stromal effects in some tumors. The identity of the perivascular BMDCs that responded to treatment is not currently known, but the intimate perivascular location of the affected cells proposes that these cells might contribute to tumor angiogenesis via paracrine mechanisms. In MCC, higher than median tumor prokineticin-2 (PROK2) mRNA content was strongly associated with the presence of Merkel cell polyomavirus (MCPyV) DNA, viral large T antigen expression, higher than median number of tumor infiltrating CD68+ and CD163+ macrophages, and with favorable survival. The presence of prokineticin-1 (PROK1) mRNA in the tumor was associated with absence of MCPyV DNA and tended to be associated with poor survival. Neither PROK1 nor PROK2 mRNA content was associated with the tumor microvascular density. Taken together, these findings support the immunomodulatory role of prokineticins and suggest that prokineticins are involved in mediating the immune response in MCC, but their role in tumor angiogenesis in MCC requires further evaluation.Syöpäkasvaimet koostuvat syöpäsolujen lisäksi vaihtelevasta määrästä erilaisia strooman soluja, joita ovat mm. verisuonien endoteelisolut ja perisyytit, fibroblastit, sekä luuydinperäiset solut. Strooman solut erittävät proteaaseja, kemokiineja, ja kasvutekijöitä muodostaen soluväliaineen kanssa syövän mikroympäristön. Mikroympäristö voi estää tai edesauttaa syöpäkasvaimen kehittymistä. Mikroympäristön tutkiminen voi tuoda uutta tietoa syövän kasvun säätelystä ja voi auttaa syövän kliinisen taudinkulun ja uusiutumisriskin arvioinnissa. Väitöskirjatyön tavoitteena oli tutkia luuydinperäisten solujen merkitystä syövän kasvussa ja verisuonien muodostuksessa. Ensimmäisessä osatyössä selvitimme verisuonien endoteelin alkuperää hiirimallien avulla. On ehdotettu, että osa verisuonien endoteelistä olisi peräisin luuydinperäisistä progenitorisoluista. Tällaisen progenitorisolun olemassaolo mahdollistaisi uuden tavan estää tai lisätä verisuonien kasvua. Tulostemme perusteella luuydin ei näyttäisi olevan merkittävässä määrin verisuonien endoteelin lähde vaan verisuonien endoteelisolut ovat peräisin jo olemassaolevista paikallisista verisuonista. Toisessa osatyössä tutkimme epidermaalisen kasvutekjän reseptorin (EGFR) merkitystä verisuonien kasvun säätelyssä hiiren syöpämallissa, jossa syöpäsolut eivät ilmennä EGFR:ää. Tulostemme perusteella EGFR osallistuu verisuonien kasvun säätelyyn, sillä sen toiminnan estäjä gefitinib vähensi kasvaimen pienien verisuonien perisyyttien määrää sekä lähellä kasvaimen verisuonia sijaitsevien luudinperäisten solujen määrää. Kolmannessa osatyössä tutkimme prokinetisiinejä, kemokiinien kaltaisia peptidejä, ihmisen harvinaisessa ihosyövässä, Merkelin solukarsinoomassa. Tulostemme perusteella prokinetisiini-2:n korkeampi määrä kasvaimessa on yhteydessä kasvaimen Merkelin polyoomaviruspositiivisuuteen (MCPyV), CD68- ja CD163-positiivisten makrofagien suurempaan määrään sekä keskimääräistä parempaan eloonjäämisennusteen. Toisen ligandin, prokinetisiini 1:n, ilmentyminen kasvaimessa on yhteydessä kasvaimen MCPyV-negatiivisuuteen, ja huonompaan eloonjäämisennusteeseen. Kummankaan ligandin määrä kasvaimessa ei korreloinut kasvaimen verisuonien määrän kanssa, joten Merkelin solukarsinoomassa prokinetisiinit näyttäisivät osallistuvan ensisijaisesti kasvaimen immuunivasteen säätelyyn

Adjuvant capecitabine-containing chemotherapy benefit and homologous recombination deficiency in early-stage triple-negative breast cancer patients

Background The addition of adjuvant capecitabine to standard chemotherapy of early-stage triple-negative breast cancer (TNBC) patients has improved survival in a few randomised trials and in meta-analyses. However, many patients did not benefit. We evaluated the BRCA1-like DNA copy number signature, indicative of homologous recombination deficiency, as a predictive biomarker for capecitabine benefit in the TNBC subgroup of the FinXX trial. Methods Early-stage TNBC patients were randomised between adjuvant capecitabine-containing (TX + CEX: capecitabine-docetaxel, followed by cyclophosphamide-epirubicin-capecitabine) and conventional chemotherapy (T + CEF: docetaxel, followed by cyclophosphamide-epirubicin-fluorouracil). Tumour BRCA1-like status was determined on low-coverage, whole genome next-generation sequencing data using an established DNA comparative genomic hybridisation algorithm. Results For 129/202 (63.9%) patients the BRCA1-like status could be determined, mostly due to lack of tissue. During a median follow-up of 10.7 years, 35 recurrences and 32 deaths occurred. Addition of capecitabine appears to improve recurrence-free survival more among 61 (47.3%) patients with non-BRCA1-like tumours (HR 0.23, 95% CI 0.08-0.70) compared to 68 (52.7%) patients with BRCA1-like tumours (HR 0.66, 95% CI 0.24-1.81) (P-interaction = 0.17). Conclusion Based on our data, patients with non-BRCA1-like TNBC appear to benefit from the addition of capecitabine to adjuvant chemotherapy. Patients with BRCA1-like TNBC may also benefit. Additional research is needed to define the subgroup within BRCA1-like TNBC patients who may not benefit from adjuvant capecitabine.Peer reviewe

Human breast cancer cells educate macrophages toward the M2 activation status

Abstract Introduction The immune system plays a major role in cancer progression. In solid tumors, 5-40 % of the tumor mass consists of tumor-associated macrophages (TAMs) and there is usually a correlation between the number of TAMs and poor prognosis, depending on the tumor type. TAMs usually resemble M2 macrophages. Unlike M1-macrophages which have pro-inflammatory and anti-cancer functions, M2-macrophages are immunosuppressive, contribute to the matrix-remodeling, and hence favor tumor growth. The role of TAMs is not fully understood in breast cancer progression. Methods Macrophage infiltration (CD68) and activation status (HLA-DRIIα, CD163) were evaluated in a large cohort of human primary breast tumors (562 tissue microarray samples), by immunohistochemistry and scored by automated image analysis algorithms. Survival between groups was compared using the Kaplan-Meier life-table method and a Cox multivariate proportional hazards model. Macrophage education by breast cancer cells was assessed by ex vivo differentiation of peripheral blood mononuclear cells (PBMCs) in the presence or absence of breast cancer cell conditioned media (MDA-MB231, MCF-7 or T47D cell lines) and M1 or M2 inducing cytokines (respectively IFN-γ, IL-4 and IL-10). Obtained macrophages were analyzed by flow cytometry (CD14, CD16, CD64, CD86, CD200R and CD163), ELISA (IL-6, IL-8, IL-10, monocyte colony stimulating factor M-CSF) and zymography (matrix metalloproteinase 9, MMP-9). Results Clinically, we found that high numbers of CD163+ M2-macrophages were strongly associated with fast proliferation, poor differentiation, estrogen receptor negativity and histological ductal type (p<0.001) in the studied cohort of human primary breast tumors. We demonstrated ex vivo that breast cancer cell-secreted factors modulate macrophage differentiation toward the M2 phenotype. Furthermore, the more aggressive mesenchymal-like cell line MDA-MB231, which secretes high levels of M-CSF, skews macrophages toward the more immunosuppressive M2c subtype. Conclusions This study demonstrates that human breast cancer cells influence macrophage differentiation and that TAM differentiation status correlates with recurrence free survival, thus further emphasizing that TAMs can similarly affect therapy efficacy and patient outcome

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Immunohistochemical analysis conditions. (PDF 273 kb

Additional file 6: of Human breast cancer cells educate macrophages toward the M2 activation status

Representative flow cytometry dot plot of IL-4 macrophages A human macrophages in the presence of IL-4 with or without breast cancer cell line conditioned media (CM), detected with antibodies against CD14, CD16, CD163, CD200R and CD86 B mean fluorescence intensity (MFI) of the analyzed surface-markers. (PDF 2715 kb

Additional file 5: of Human breast cancer cells educate macrophages toward the M2 activation status

Representative flow cytometry dot plot of IL-10 macrophages A human macrophages in the presence of IL-10 with or without breast cancer cell line conditioned media (CM), detected with antibodies against CD14, CD16 and CD163 B mean fluorescence intensity (MFI) of the analyzed surface-markers. (PDF 18647 kb