Innate Immune Cells: Mediators of the Angiogenic Switch?

The role of Matrix metalloprotease type 9 (MMP-9) in the activation of Vascular Endothelial Growth Factor (VEGF), the induction and maintenance of chronic angiogenesis and early stage tumor growth has been well established. But what is the source of MMP-9 ? In a study published in 2006, Nozawa and co-workers, using the RIP1-Tag2 transgenic mouse as a model, identified two inflammatory cell types, neutrophils and macrophages, as the major sources of MMP-9.

The RIP1-Tag2 transgenic mouse is a well characterized model of multistep carcinogenesis involving the pancreatic islets. Nozawa and co-workers first isolated constituent cell types from a tumor and cell-sorted Gr-1⁺ and Mac-1⁺ cells and then carried out a semi-quantitative RT-PCR (see Fig.1)

Fig. 1: Semi-quantitative RT-PCR. MMP-9⁺ is expressed by Gr-1⁺ and Mac-1⁺cells innate immune cells (Adapted from Nozawa et. al, 2006)

Clearly, MMP-9 was expressed predominantly by innate immune cells positive for the Gr-1 and Mac-1 markers.

In order to determine the localization of these cells , a double label immunohistochemical staining of various leukocyte markers in normal and neoplastic tissue was performed. CD68 and F4/80 are two macrophage markers. Cells positive for both markers were observed inside as well as on the periphery of angiogenic islets and tumors (see Fig. 2). However, the cells inside were MMP-9^- while those along the periphery were MMP-9⁺ 

Fig. 2: MMP-9⁺ macrophages located along the periphery of the angiogenic islet.Macrophages inside the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Along similar lines, an antibody against the "7/4" (a neutrophil marker) was used to localize neutrophils. Suprisingly, the situation, this time, was the opposite. 7/4⁺ cells inside the lesions were also MMP-9⁺ while 7/4⁺ cells along the periphery were not (see Fig. 3). All  7/4⁺ cells also displayed polymorphic nuclei, strongly indicating that these cells were neutrophils.

Fig. 3: MMP-9⁺ neutrophils located inside the angiogenic islet. Neutrophils along the periphery of the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Neutrophils (7/4⁺ and Gr-1⁺) represented only 0.4% of the total tumor cells and expressed high levels of MMP-9. In contrast, macrophages (CD68⁺) represented 2% of the total tumor cells and expressed low levels of MMP-9.

The researchers then tried to determine the role of these MMP-9⁺ Gr-1⁺ cells that infiltrate angiogenic islets and tumors. An experimental regimen involving daily inoculation of anti-Gr-1 at 7 weeks (when hyperplastic islets start to undergo angiogenesis) was followed.

After 1 week of injection, most neutrophils disappeared from the pancreatic islets of the transgenic mice (see Fig.4)

Fig. 4: Left: Control; Right: Following 1 week of treatment with anti-Gr-1, MMP-9⁺ neutrophils located inside the angiogenic islet disappeared (Adapted from Nozawa et. al, 2006)

After two weeks of injection, the neutrophil population rebounded in neoplastic islets. Incredibly, none of the rebound neutrophils were MMP-9⁺ (see Fig. 5)!

Fig. 5: Left: Control; Right: Following 2 weeks of treatment with anti-Gr-1, neutrophils rebounded back into the islets. However, none of the rebound neutrophils were MMP-9⁺  (Adapted from Nozawa et. al, 2006)

In a short 2 week prevention trial (week7-week9), carried out to assess angiogenic switching frequency, the anti-Gr-1 antibody regimen, reduced the number of angiogenic islets by 57%!

Furthermore, immunostaining for VEGF:VEGF-R2 complex with GVM39 (red) and for endothelial cells with Meca-32 (green) showed a decrease in bioactive VEGF:VEGF-R2 interaction, consequent to the depletion of infiltrating MMP-9⁺ neutrophils (see Fig.6)

Fig. 6: Left: Control; Right: Short intervention trials lead to reduced VEGF:VEGF-R2 interactions. Endothelial cells are stained with an antibody directed against Meca-32 (a pan-endothelial cell marker)  (Adapted from Nozawa et. al, 2006)

Taken together, the results from this paper indicate that subtle infiltration by innate immune cells (such as neutrophils) may play a role in the progression of neoplasias towards angiogenic tumors.

Reference:

Nozawa H, Chiu C, & Hanahan D (2006). Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 103 (33), 12493-8 PMID: 16891410

Evidence for Systemic Spread prior to the Establishment of Primary Tumors

It is widely believed that metastasis is a late event in cancer progression. This view is based on several clinical and experimental observations. First, most cancer patients die from metastasis and not the primary disease. Second, early surgery is often the only cure. Third, somatic genetic changes accumulate during local progression (Fearon and Vogelstein, 1990) which was extrapolated to systemic progression. Fourth, repeated rounds of in vivo selection led to cell lines with increased metastasis formation (Kang et. al 2003; Minn et. al 2005). However, in a recent study titled Systemic Spread is an Early Step in Breast Cancer, published in Cell in 2008, Hüsemann, Geigl, Schubert et. al report that tumor cells can disseminate systemically from the earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. 

The mouse model they work with is the BALB/c mice transgenic for the activated rat HER-2/neu gene (BALB-Neu T mice). This model mimics progression and gene expression profiles of human breast cancer. Females hemizygous for the rat HER-2 gene under control of the MMTV (mouse mammary tumor virus) promoter develop  invasive mammary cancer while their HER-2 negative siblings (wild-type BALB/c mice) remain tumor free. Typically, in the BALB-Neu T model, the mammary epithelia starts to express the oncogene at about weeks 3 to 4 (which coincides with the onset of puberty). Hyperplasia can be detected microscopically at weeks 7-9. Progress to in situ  carcinomas occurs between weeks 14-18 and at weeks 23-30, invasive cancers become apparent (see Fig. 1a)

Fig. 1a; Left Panel: Whole mount of the mammary gland at week 9 showing absence of tumor in branching ductal tree; Middle Panel: Histological Section at week 9 showing side buds displaying the morphology of atypical ductal hyperplasia; Right Panel: Histology of invasive cancer at week 30 (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

The principle goal was to determine when cells expressing the HER-2 transgene disseminate. The authors chose the lung and bone marrow as the sites to look out for metastasis since the HER-2 receptor is not expressed in either of these two organs. In addition to HER, they also used anti-cytokeratin (CK) antibodies to check whether disseminated cells were epithelial in origin. Surprisingly in BALB-Neu T mice, CK⁺ and HER-2⁺ cells became detectable 4-9 weeks when meticulous analysis could only detect atypical ductal hyperplasia (ADH) (see Fig. 1b) 

Fig. 1b; Left Panel: Increase in tumor area at primary site. Triangles indicate mean value, whiskers indicate 95% confidence interval and solid line indicates best fitted curve; Right Panel: Number of CK⁺ cells (red dots) and HER⁺ cells (blue dots) per 5000 bone marrow cells. Triangles, whiskers and solid line indicate same. Note the presence of disseminated cells in the bone marrow even when the primary tumor area is zero.(Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Further, lung micrometastasis from mammary tissue was confirmed by demonstration of mammary-specific alpha casein and lactalbumin transcripts (see Fig. 1c)

Fig. 1c; Left Panel: Lung micrometastasis at week 27, detected using anti-HER-2 antibody;Right Panel: Eight HER-2 ⁺ and two samples of normal lung tissues, analyzed for mammary gland specific transcripts. "+" indicates normal mammary gland and "-" indicates mock control (Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Despite exponential growth at the primary tumor site, the number of CK⁺ cells and HER-2⁺cells in the bone marrow rose marginally over the course of time. Cells singly positive for HER-2 and CK were not congruent. Not all tumor cells expressed both markers (see Fig. 1d)

Fig. 1d; Left Panel: CK⁺, HER-2^- cell; Middle Panel: CK^-, HER-2⁺ cell; Right Panel: CK⁺, HER-2⁺ cell (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

This suggests, either the existence of heterogeneous tumor cell populations that disseminate to distant sites or different cellular states of the disseminated tumor cells (DTCs)

More Evidence for Early Dissemination

Could the HER-2⁺, CK⁺ cells have disseminated from extra-mammary tissues expressing the transgene? The authors took mammary gland fragments from 3-12 week old transgenic mice (displaying only atypical ductal hyperplasia) and transplanted the them into 3 week old wild-type siblings.  Wild type bone marrow was screened at different time points (see Fig. 2)

Fig. 2; Left Panel: Week 14 post-transplantation, CK⁺ cells in bone marrow of wild-type siblings; Right Panel: HER-2⁺ cells in the bone marrow (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Although CK⁺ and HER-2⁺ cells were observed in the bone marrow, the levels were lower than transgenic BALB-Neu T mice, but were definitely above rare false positives. Additionally, just as in the transgenic mice, there was no significant increase in disseminated cells from ADH stages to invasive cancer, in the wild-type siblings. 

The malignant nature of these DTCs was established by Comparative Genomics Hybridization (see Fig. 3)

Fig. 3; Top Panel: Karyogram (left) and CGH profile (right) of sorted mouse metaphase of a single HER-2⁺ cell disseminated from a transplanted mammary gland and subsequently isolated from bone marrow of a recipient wild-type mouse; Bottom Panel: Sorting of mouse metaphase of a single leukocyte in a balanced CGH profile; Green and red bars indicate genomic gains and losses respectively. (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Evidence that early disseminated cells can grow into metastases

The onset of metastasis relative to primary tumor growth was assessed . Histological sections of the lungs were analyzed and micrometastases could be detected from weeks 20 to 21 onward, a time point at which mostly in-situ carcinomas are present at the primary sites (see Fig. 4)

Fig. 4; Progression of lung metastasis in BALB-Neu T mice, with and without the removal of the primary tumor. Left Panel: Increase in tumor area over time in BALB-Neu T mice (same as Fig. 1b); Right Panel: Size of the largest lung metastasis detected in individual mice. Blue squares indicate average size of metastases from non-operated mice at various time points and red triangles indicate average size from operated animals at 10-15 weeks after surgery. Whiskers indicate 95% confidence intervals (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Since metastases need time to grow, their increase in size paralleling that of the primary lesion supports the conclusion that, at least in some cases, founder cells of metastasis had disseminated earlier and had started to proliferate.

Some other experiments conducted by the group, demonstrate that bone marrow disseminated cells that do not grow into metastases can be released from growth arrest. Also, there does not appear to be an association between the number of disseminated cells and the stage of the tumor. Large tumors do not necessarily seed more.

Overall the paper presents some compelling evidence for the occurrence of dissemination, even before the primary tumor is established. This would indeed force the field to rethink it's current strategies in combating cancer.

Reference:

Hüsemann, Y., Geigl, J., Schubert, F., Musiani, P., Meyer, M., Burghart, E., Forni, G., Eils, R., Fehm, T., & Riethmüller, G. (2008). Systemic Spread Is an Early Step in Breast Cancer Cancer Cell, 13 (1), 58-68 DOI: 10.1016/j.ccr.2007.12.003