Rat prostate tumors induce DNA synthesis in remote organs.

Advanced cancers induce systemic responses. However, if such systemic changes occur already when aggressive tumors are small, have not been thoroughly characterized. Here, we examined how localized prostate cancers of different sizes and metastatic potential affected DNA synthesis in the rest of the prostate and in various remote organs. Non-metastatic Dunning R-3327 G (G) tumor cells, metastatic MatLyLu (MLL) tumor cells, or vehicle were injected into the prostate of immunocompetent rats. All animals received daily injections of Bromodeoxyuridine (BrdU), to label cells/daughter cells with active DNA synthesis. Equal sized G- and MLL-tumors, similarly increased BrdU-labeling in the prostate, lymph nodes and liver compared to tumor-free controls. Prior to metastasis, MLL-tumors also increased BrdU-labeling in bone marrow and lungs compared to animals with G-tumors or controls. In animals with MLL-tumors, BrdU-labeling in prostate, lungs, brown adipose tissue and skeletal muscles increased in a tumor-size-dependent way. Furthermore, MLL-tumors induced increased signs of DNA damage (γH2AX staining) and accumulation of CD68 + macrophages in the lungs. In conclusion, small localized prostate cancers increased DNA synthesis in several remote tissues in a tumor type- and size-dependent way. This may suggest the possibility for early diagnosis of aggressive prostate cancer by examining tumor-induced effects in other tissues.

High-grade tumours promote growth of other less-malignant tumours in the same prostate.

Prostate cancer is a multifocal disease, but if and how individual prostate tumours influence each other is largely unknown. We therefore explored signs of direct or indirect tumour-tumour interactions in experimental models and patient samples. Low-metastatic AT1 and high-metastatic MatLyLu (MLL) Dunning rat prostate cancer cells were injected into separate lobes of the ventral prostate of immunocompetent rats. AT1 tumours growing in the same prostate as MLL tumours had increased tumour size and proliferation compared to AT1 tumours growing alone. In addition, the vasculature and macrophage density surrounding the AT1 tumours were increased by MLL tumour closeness. In patient prostatectomy samples, selected to contain an index tumour [tumour with the highest grade, International Society of Urological Pathology (ISUP) grade 1, 2, 3 or 4] and a low-grade satellite tumour (ISUP grade 1), cell proliferation in low-grade satellite tumours gradually increased with increasing histological grade of the index tumour. The density of blood vessels and CD68+ macrophages also increased around the low-grade satellite tumour if a high-grade index tumour was present. This suggests that high-grade tumours, by changing the prostate microenvironment, may increase the aggressiveness of low-grade lesions in the organ. Future studies are needed to explore the mechanisms behind tumour-tumour interactions and their clinical importance. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Prostate cancer induces C/EBPß expression in surrounding epithelial cells which relates to tumor aggressiveness and patient outcome.

BACKGROUND: Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating adaptations in the tumor-bearing organ. Similar changes are seen in prostate cancer patients and they are related to outcome. One gene previously found to be upregulated in the non-malignant part of tumor-bearing prostate lobe in rats was the transcription factor CCAAT/enhancer-binding protein-ß (C/EBPß). METHODS: To explore this further, we examined C/EBPß expression by quantitative RT-PCR, immunohistochemistry, and Western blot in normal rat prostate tissue surrounding slow-growing non-metastatic Dunning G, rapidly growing poorly metastatic (AT-1), and rapidly growing highly metastatic (MatLyLu) rat prostate tumors-and also by immunohistochemistry in a tissue microarray (TMA) from prostate cancer patients managed by watchful waiting. RESULTS: In rats, C/EBPß mRNA expression was upregulated in the surrounding tumor-bearing prostate lobe. In tumors and in the surrounding non-malignant prostate tissue, C/EBPß was detected by immunohistochemistry in some epithelial cells and in infiltrating macrophages. The magnitude of glandular epithelial C/EBPß expression in the tumor-bearing prostates was associated with tumor size, distance to the tumor, and metastatic capacity. In prostate cancer patients, high expression of C/EBPß in glandular epithelial cells in the surrounding tumor-bearing tissue was associated with accumulation of M1 macrophages (iNOS+) and favorable outcome. High expression of C/EBPß in tumor epithelial cells was associated with high Gleason score, high tumor cell proliferation, metastases, and poor outcome. CONCLUSIONS: This study suggest that the expression of C/EBP-beta, a transcription factor mediating multiple biological effects, is differentially expressed both in the benign parts of the tumor-bearing prostate and in prostate tumors, and that alterations in this may be related to patient outcome.

Highly aggressive rat prostate tumors rapidly precondition regional lymph nodes for subsequent metastatic growth.

The aim of this study was to examine in what ways MatLyLu (MLL) rat prostate tumors with high metastatic capacity influence regional lymph nodes prior to metastatic establishment compared to AT1 rat prostate tumors with low metastatic potential. MLL or AT1 tumor cells were injected into the ventral prostate of immunocompetent rats. Tumor and lymph node morphology, and lymph node mRNA expression of macrophage associated markers, T-cell associated markers, and cytokines were examined over time until the first microscopic signs of metastases (at day 14 for MLL- and at day 28 for AT1-tumors). Already at day 3 after tumor cell injection, when the tumors were extremely small and occupied less than 1% of the prostate volume, MLL- and AT1-tumors provoked different immune responses in both the prostate and the regional lymph nodes. MLL-tumors induced expression of immunosuppressive cytokines, suppressed T-cell accumulation, and directed T-cells towards an immunosuppressive phenotype. AT1-tumors caused a response more similar to that in vehicle-injected animals, with accumulation of T-cells in tumors and regional lymph nodes. Prostate tumors with high metastatic potential were able to precondition regional lymph nodes to subsequent metastatic growth in ways different from tumors with less metastatic potential. This may indicate the existence of a time-window when pre-metastatic changes in regional lymph nodes can aid in the prognostication of locally aggressive and potentially metastatic prostate cancer.

Reduced number of CD169+ macrophages in pre-metastatic regional lymph nodes is associated with subsequent metastatic disease in an animal model and with poor outcome in prostate cancer patients.

BACKGROUND: Tumor-derived antigens are captured by CD169+ (SIGLEC1+ ) sinus macrophages in regional lymph nodes (LNs), and are presented to effector cells inducing an anti-tumor immune response. Reduced CD169 expression in pre-metastatic regional LNs is associated with subsequent metastatic disease and a poor outcome in several tumor types, but if this is the case in prostate cancer has not been explored. METHODS: CD169 expression was measured with immunohistochemistry in metastasis-free regional LNs from 109 prostate cancer patients treated with prostatectomy (January 1996 to April 2002). Possible associations of CD169 expression with PSA-relapse, prostate cancer death, Gleason score, and other clinical data were assessed using Kaplan-Meier survival- and Cox regression analysis. In addition, the Dunning rat prostate tumor model was used to examine CD169 expression in pre-metastatic LNs draining either highly metastatic MatLyLu- or poorly metastatic AT1-tumors. RESULTS: In patients with low CD169 immunostaining in metastasis-free regional LNs, 8 of the 27 patients died from prostate cancer compared with only three of the 82 patients with high immunostaining (P < 0.001). CD169 expression in regional LNs was not associated with PSA-relapse. Rats with highly metastatic tumors had decreased CD169 immunoreactivity in pre-metastatic regional LNs compared with rats with poorly metastatic tumors. CONCLUSION: Low expression of CD169 in metastasis-free regional LNs indicates a reduced anti-tumor immune response. If verified in other studies, CD169 expression in regional LNs could, in combination with other factors, potentially be used as a marker of prostate cancer aggressiveness.

Aggressive rat prostate tumors reprogram the benign parts of the prostate and regional lymph nodes prior to metastasis.

In order to grow and spread tumors need to interact with adjacent tissues. We therefore hypothesized that small but aggressive prostate cancers influence the rest of the prostate and regional lymph nodes differently than tumors that are more indolent. Poorly metastatic (Dunning AT1) or highly metastatic (Dunning MLL) rat prostate tumor cells were injected into the ventral prostate lobe of immunocompetent rats. After 10 days-when the tumors occupied about 30% of the prostate lobe and lymph node metastases were undetectable-the global gene expression in tumors, benign parts of the prostate, and regional iliac lymph nodes were examined to define tumor-induced changes related to preparation for future metastasis. The tumors induced profound effects on the gene expression profiles in the benign parts of the prostate and these were strikingly different in the two tumor models. Gene ontology enrichment analysis suggested that tumors with high metastatic capacity were more successful than less metastatic tumors in inducing tumor-promoting changes and suppressing anti-tumor immune responses in the entire prostate. Some of these differences such as altered angiogenesis, nerve density, accumulation of T-cells and macrophages were verified by immunohistochemistry. Gene expression alterations in the regional lymph nodes suggested decreased quantity and activation of immune cells in MLL-lymph nodes that were also verified by immunostaining. In summary, even when small highly metastatic prostate tumors can affect the entire tumor-bearing organ and pre-metastatic lymph nodes differently than less metastatic tumors. When the kinetics of these extratumoral influences (by us named TINT = tumor instructed normal tissue) are more precisely defined they could potentially be used as markers of disease aggressiveness and become therapeutic targets.

Extracellular Vesicles from Metastatic Rat Prostate Tumors Prime the Normal Prostate Tissue to Facilitate Tumor Growth.

Accumulating data indicates that tumor-derived extracellular vesicles (EVs) are responsible for tumor-promoting effects. However, if tumor EVs also prepare the tumor-bearing organ for subsequent tumor growth, and if this effect is different in low and high malignant tumors is not thoroughly explored. Here we used orthotopic rat Dunning R-3327 prostate tumors to compare the role of EVs from fast growing and metastatic MatLyLu (MLL) tumors with EVs from more indolent and non-metastatic Dunning G (G) tumors. Prostate tissue pre-conditioned with MLL-EVs in vivo facilitated G tumor establishment compared to G-EVs. MLL-EVs increased prostate epithelial proliferation and macrophage infiltration into the prostate compared to G-EVs. Both types of EVs increased macrophage endocytosis and the mRNA expression of genes associated with M2 polarization in vitro, with MLL-EVs giving the most pronounced effects. MLL-EVs also altered the mRNA expression of growth factors and cytokines in primary rat prostate fibroblasts compared to G-EVs, suggesting fibroblast activation. Our findings propose that EVs from metastatic tumors have the ability to prime the prostate tissue and enhance tumor growth to a higher extent than EVs from non-metastatic tumors. Identifying these differences could lead to novel therapeutic targets and potential prognostic markers for prostate cancer.

Extratumoral Heme Oxygenase-1 (HO-1) Expressing Macrophages Likely Promote Primary and Metastatic Prostate Tumor Growth.

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

Inhibition of Lysyl Oxidase and Lysyl Oxidase-Like Enzymes Has Tumour-Promoting and Tumour-Suppressing Roles in Experimental Prostate Cancer.

Lysyl oxidase (LOX) and LOX-like (LOXL) enzymes are key players in extracellular matrix deposition and maturation. LOX promote tumour progression and metastasis, but it may also have tumour-inhibitory effects. Here we show that orthotopic implantation of rat prostate AT-1 tumour cells increased LOX and LOXLs mRNA expressions in the tumour and in the surrounding non-malignant prostate tissue. Inhibition of LOX enzymes, using Beta-aminopropionitrile (BAPN), initiated before implantation of AT-1 cells, reduced tumour growth. Conversely, treatment that was started after the tumours were established resulted in unaffected or increased tumour growth. Moreover, treatment with BAPN did not suppress the formation of spontaneous lymph node metastases, or lung tumour burden, when tumour cells were injected intravenously. A temporal decrease in collagen fibre content, which is a target for LOX, was observed in tumours and in the tumour-adjacent prostate tissue. This may explain why early BAPN treatment is more effective in inhibiting tumour growth compared to treatment initiated later. Our data suggest that the enzymatic function of the LOX family is context-dependent, with both tumour-suppressing and tumour-promoting properties in prostate cancer. Further investigations are needed to understand the circumstances under which LOX inhibition may be used as a therapeutic target for cancer patients.

Adaptive (TINT) Changes in the Tumor Bearing Organ Are Related to Prostate Tumor Size and Aggressiveness.

In order to grow, tumors need to induce supportive alterations in the tumor-bearing organ, by us named tumor instructed normal tissue (TINT) changes. We now examined if the nature and magnitude of these responses were related to tumor size and aggressiveness. Three different Dunning rat prostate tumor cells were implanted into the prostate of immune-competent rats; 1) fast growing and metastatic MatLyLu tumor cells 2) fast growing and poorly metastatic AT-1 tumor cells, and 3) slow growing and non-metastatic G tumor cells. All tumor types induced increases in macrophage, mast cell and vascular densities and in vascular cell-proliferation in the tumor-bearing prostate lobe compared to controls. These increases occurred in parallel with tumor growth. The most pronounced and rapid responses were seen in the prostate tissue surrounding MatLyLu tumors. They were, also when small, particularly effective in attracting macrophages and stimulating growth of not only micro-vessels but also small arteries and veins compared to the less aggressive AT-1 and G tumors. The nature and magnitude of tumor-induced changes in the tumor-bearing organ are related to tumor size but also to tumor aggressiveness. These findings, supported by previous observation in patient samples, suggest that one additional way to evaluate prostate tumor aggressiveness could be to monitor its effect on adjacent tissues.

High Lysyl Oxidase (LOX) in the Non-Malignant Prostate Epithelium Predicts a Poor Outcome in Prostate Cancer Patient Managed by Watchful Waiting.

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.

Characterization of a Gene Expression Signature in Normal Rat Prostate Tissue Induced by the Presence of a Tumor Elsewhere in the Organ.

Implantation of rat prostate cancer cells into the normal rat prostate results in tumor-stimulating changes in the tumor-bearing organ, for example growth of the vasculature, an altered extracellular matrix, and influx of inflammatory cells. To investigate this response further, we compared prostate morphology and the gene expression profile of tumor-bearing normal rat prostate tissue (termed tumor-instructed/indicating normal tissue (TINT)) with that of prostate tissue from controls. Dunning rat AT-1 prostate cancer cells were injected into rat prostate and tumors were established after 10 days. As controls we used intact animals, animals injected with heat-killed AT-1 cells or cell culture medium. None of the controls showed morphological TINT-changes. A rat Illumina whole-genome expression array was used to analyze gene expression in AT-1 tumors, TINT, and in medium injected prostate tissue. We identified 423 upregulated genes and 38 downregulated genes (p<0.05, ≥2-fold change) in TINT relative to controls. Quantitative RT-PCR analysis verified key TINT-changes, and they were not detected in controls. Expression of some genes was changed in a manner similar to that in the tumor, whereas other changes were exclusive to TINT. Ontological analysis using GeneGo software showed that the TINT gene expression profile was coupled to processes such as inflammation, immune response, and wounding. Many of the genes whose expression is altered in TINT have well-established roles in tumor biology, and the present findings indicate that they may also function by adapting the surrounding tumor-bearing organ to the needs of the tumor. Even though a minor tumor cell contamination in TINT samples cannot be ruled out, our data suggest that there are tumor-induced changes in gene expression in the normal tumor-bearing organ which can probably not be explained by tumor cell contamination. It is important to validate these changes further, as they could hypothetically serve as novel diagnostic and prognostic markers of prostate cancer.