First report of fig mosaic virus on fig in Russia.

Fig mosaic virus (FMV) (genus Emaravirus in the family Fimoviridae) is considered the etiological agent of fig mosaic disease (FMD) that is recorded in most of the fig growing areas with an average global infection rate of 33%. The multipartite FMV genome is comprised of six negative monocistronic ssRNAs, each of which is separately encapsidated (Preising et al. 2020). Although FMD-like symptoms, which include mosaic, chlorotic ringspots, and oak leaf patterns, were observed in approximately a third of 400 fig accessions in the Nikita Botanical Gardens, Yalta, Russia (Mitrofanova et al. 2016), FMV has not been identified as the causal agent of the disease. In June of 2020, total RNA was isolated from symptomatic leaves of 59 thirty two-year-old trees representing 31 local and 27 introduced Ficus carica L. cultivars and a single F. pseudocarica Miq. tree using RNeasy Plant Mini kit (Qiagen, USA). FMV was tested by RT-PCR using primer sets E5 (Elbeaino et al. 2009) and EMARAVGP (Walia et al. 2009), which amplify a 302-bp fragment of RNA1 and a 468-bp fragment of RNA2, respectively. PCR products of the expected sizes were generated in all samples, indicating a high FMV incidence in the plantings. The genome sequences of FMV isolates from F. carica cvs. Bleuet, Kraps di Hersh, Smena, Temri, and F. pseudocarica (Fig. S1) were determined by high-throughput sequencing on MiSec Illumina platform. Double-stranded RNA was isolated from FMV-positive leaves using Viral Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, USA). In average, 695,000 quality-filtered 150 bp pair-ended reads per a library were produced and used in a de novo assembly using metaSpades program version 3.14 (Nurk et al. 2017). In each of five samples, BLASTn analysis found six FMV-related contigs. The contigs spanned 99 to 100% of corresponding genomic segments of the most closely related isolates. In addition to FMV, fig cryptic virus-related contigs were also detected in some samples. The FMV contigs covering RNA1 to RNA6 had the highest identity to corresponding genomic segments of isolates AM941711 (96.5 to 96.6%), FM864225 (94.4 to 94.6%), FM991954 (97.9 to 98.2%), AB697863 (96.4 to 96.6%), AB697879 (93.3 to 93.4%), and AB697895 (95.4 to 97.0%), respectively. Five Russian isolates shared 99.2 to 100% nucleotide sequence identity, depending on the genomic segment. Their sequences were deposited in GenBank under accession numbers MW201216 to MW201230 and MW208662 to MW208676. Phylogenetic analysis of six ORFs showed that ORF1 to ORF3 and ORF6 of the Russian isolates clustered with FMV isolates from Italy while ORF4 grouped with the isolate JTT-Pa (AB697863) from Japan (Fig. S2). ORF5 of the Russian isolates formed a separate cluster with the isolates SB1 and SB2 from Serbia and JTT-Vi from Japan (AB697879 to AB697884). Incongruency of phylogenetic relationship among the genomic segments suggests reassortment among ancestors of the Russian FMV isolates. In addition, similar to the SB1, SB2 and JTT-Vi, ORF5 of the Russian isolates encodes a protein of 486 amino acid (aa) residues in contrast to the corresponding protein of Italian isolates consisting of 502 aa. To the best of our knowledge, this is the first report of FMV in Russia. This finding not only expands the information on the geographical distribution of FMV, but also extends knowledge on F. pseudocarica as a natural host of the virus.

Molecular characterization of Plum pox virus Rec isolates from Russia suggests a new insight into evolution of the strain.

Field isolates of Plum pox virus (PPV), belonging to the strain Rec, have been found for the first time in Russia. Full-size genomes of the isolates K28 and Kisl-1pl from myrobalan and plum, respectively, were sequenced on the 454 platform. Analysis of all known PPV-Rec complete genomes using the Recombination Detection Program (RDP4) revealed yet another recombination event in the 5'-terminal region. This event was detected by seven algorithms, implemented in the RDP4, with statistically significant P values and supported by a phylogenetic analysis with the bootstrap value of 87%. A putative PPV-M-derived segment, encompassing the C-terminus of the P1 gene and approximately two-thirds of the HcPro gene, is bordered by breakpoints at positions 760-940 and 1838-1964, depending on the recombinant isolate. The predicted 5'-distal breakpoint for the isolate Valjevka is located at position 2804. The Dideron (strain D) and SK68 (strain M) isolates were inferred as major and minor parents, respectively. Finding of another recombination event suggests more complex evolutionary history of PPV-Rec than previously assumed. Perhaps the first recombination event led to the formation of a PPV-D variant harboring the PPV-M-derived fragment within the 5'-proximal part of the genome. Subsequent recombination of its descendant with PPV-M in the 3'-proximal genomic region resulted in the emergence of the evolutionary successful strain Rec.

Role of chitinase-like proteins in cancer.

Chitinase-like proteins (CLPs) are lectins combining properties of cytokines and growth factors. Human CLPs include YKL-40, YKL-39 and SI-CLP that are secreted by cancer cells, macrophages, neutrophils, synoviocytes, chondrocytes and other cells. The best investigated CLP in cancer is YKL-40. Serum and plasma levels of YKL-40 correlate with poor prognosis in breast, lung, prostate, liver, bladder, colon and other types of cancers. In combination with other circulating factors YKL-40 can be used as a predictive biomarker of cancer outcome. In experimental models YKL-40 supports tumor initiation through binding to RAGE, and is able to induce cancer cell proliferation via ERK1/2-MAPK pathway. YKL-40 supports tumor angiogenesis by interaction with syndecan-1 on endothelial cells and metastatic spread by stimulating production of pro-inflammatory and pro-invasive factors MMP9, CCL2 and CXCL2. CLPs induce production of pro- and anti-inflammatory cytokines and chemokines, and are potential modulators of inflammatory tumor microenvironment. Targeting YKL-40 using neutralizing antibodies exerts anti-cancer effect in preclinical animal models. Multifunctional role of CLPs in regulation of inflammation and intratumoral processes makes them attractive candidates for tumor therapy and immunomodulation. In this review we comprehensively analyze recent data about expression pattern, and involvement of human CLPs in cancer.

Occurrence and characterization of plum pox virus strain D isolates from European Russia and Crimea.

Numerous plum pox virus (PPV) strain D isolates have been found in geographically distant regions of European Russia and the Crimean peninsula on different stone fruit hosts. Phylogenetic analysis of their partial and complete genomes suggests multiple introductions of PPV-D into Russia. Distinct natural isolates from Prunus tomentosa were found to bear unique amino acid substitutions in the N-terminus of the coat protein (CP) that may contribute to the adaptation of PPV-D to this host. Serological analysis using the PPV-D-specific monoclonal antibody 4DG5 provided further evidence that mutations at positions 58 and 59 of the CP are crucial for antibody binding.

First Report and Molecular Characterization of Umbra-like Virus on Ficus carica Caprifig Trees in Crimea.

Fig mosaic is the most serious viral disease affecting figs. A fig germplasm collection from the Nikita Botanical Garden on the Crimean Peninsula was surveyed for viruses using high-throughput sequencing and RT-PCR with primers specific to known fig viruses. Reads related to fig umbra-like virus (FULV) were generated in samples from Ficus carica caprifig (pollinator) trees of the cultivar Belle dure. F. carica trees of other cultivars, as well as F. afghanistanica, F. palmata, and F. virgata trees, tested negative for FULV. Near-complete genomes of five Crimean fig umbra-like virus (FULV-CR) isolates shared 99.4% to 99.9% identity and were most closely related (85.2% identity) to the Hawaiian FULV isolate Oahu1 (MW480892). Based on their genome structure and a phylogenetic analysis, the FULV-CR isolates were determined to be dicot-infecting Class 2 umbra-like viruses and seem to be highly divergent forms of the same virus found recently in Hawaii, USA. This is the first report of an umbra-like virus found on figs in Crimea and outside of Hawaii, expanding information on the geographical distribution and genetic diversity of FULV. All of the Crimean FULV-positive plants were also co-infected with fig mosaic virus, fig badnavirus 1, and grapevine badna FI virus.

First Report and Complete Genome Characterization of Cherry Virus A and Little Cherry Virus 1 from Russia.

Virus diseases affect the yield and fruit quality and shorten the productive life of stone fruits (Prunus spp. in the family Rosaceae). Of over fifty known viruses infecting these crops, cherry virus A (CVA) is among the most common, and little cherry virus 1 (LChV1) is one of the most economically important. Using high-throughput sequencing, full-length genomes of CVA and LChV1 isolates, found on interspecies hybrids in the Prunus collection of the Nikita Botanical Gardens, Russia, were sequenced, assembled, and characterized. CVA was found in the P. cerasifera × P. armeniaca hybrid and in phylogenetic analysis clustered with non-cherry virus isolates. The LChV1 isolate Stepnoe was detected in ((P. cerasifera Ehrh. × P. armeniaca L.) × P. brigantiaca Vill.) trihybrid suggesting that both P. cerasifera and P. brigantiaca potentially can be the LChV1 hosts. The isolate Stepnoe was most closely related to the Greece isolate G15_3 from sweet cherry, sharing 77.3% identity at the nucleotide level. Possibly, the highly divergent Russian isolate represents one more phylogroup of this virus. This is the first report of CVA and LChV1 from Russia, expanding the information on their geographical distribution and genetic diversity.

Characterization of Divergent Grapevine Badnavirus 1 Isolates Found on Different Fig Species (Ficus spp.).

Fig mosaic disease is spread worldwide and is believed to have a viral etiology. Divergent isolates of grapevine badnavirus 1 (GBV1), named fGBV1, were discovered on Ficus carica, F. palmata, F. virgata, and F. afghanistanica in the fig germplasm collection of the Nikita Botanical Gardens, Russia, expanding the list of viruses infecting this crop. The complete genomes of five fGBV1 isolates from F. carica and F. palmata trees were determined using high-throughput and Sanger sequencing. The genomes comprised 7283 base pairs, contained four overlapping open reading frames, were 99.7 to 99.9% identical to each other, and related to GBV1 (83.2% identity). The reverse transcriptase RNase H genome regions of fGBV1 and GBV1 share 84.6% identity, indicating that fGBV1 is a divergent isolate of GBV1, which was found on the new natural hosts from a different family (Moraceae). Further, fGBV1-specific primers were developed to detect the virus using RT-PCR. Survey of 47 trees, belonging to four fig species and 14 local and introduced F. carica cultivars, showed the high fGBV1 prevalence in the collection (93.6%), including trees with no obvious symptoms of fig mosaic disease.

Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia.

BACKGROUND: Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. METHODS: We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. RESULTS: A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. CONCLUSION: We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.

A de novo genome assembly of cultivated Prunus persica cv. 'Sovetskiy'.

Prunus persica is one of the main stone fruit crops in Crimea and southern Russia. The P. persica genome has recently been sequenced and annotated in good quality. However, for a deeper assessment of the peach genome, it is necessary to include in the research other cultivars that are in the collection of the Nikitsky Botanical Garden. The cultivars of the Nikitsky Botanical Garden are unique and differ from Western European and American ones, as they are derived from cultivars and forms originating from Central Asian, North Caucasian, Transcaucasian and Eastern European countries. In this paper, we present the assembly of the P. persica cv. 'Sovetskiy' genome obtained using Oxford Nanopore long reads and Illumina short reads by hybrid assembly methods. The assembled genome of P. persica cv. 'Sovetskiy' is 206.26 MB in 226 scaffolds, with N50 24 Mb, including 8 chromosomes. It contains 27140 coding genes, 26973 (99.38%) of which are annotated in at least one functional database. More than 36.05% of the genome regions were identified as repeating elements.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars 'Alpinist,' 'Ay-Nor,' 'Bal Tsvetov,' 'Crimson Star,' 'Crystal Fountain,' 'Kosmicheskaya Melodiya,' 'Lesnaya Opera,' 'Madame Julia Correvon,' 'Nevesta,' 'Nikitsky Rosovyi,' 'Nikolay Rubtsov,' 'Serenada Kryma,' and 'Vechniy Zov' were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3-0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20-8.90 µM) and 0.049 µM NAA, or TDZ (3.0; 6.0, and 9.0 µM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 µM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 µmol m-2 s-1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 µM BAP, or 6.0 µM TDZ were optimal cytokinin concentrations for micropropagation. The explants of 'Alpinist,' 'Ay-Nor,' 'Crimson Star,' 'Crystal Fountain,' 'Nevesta,' and 'Serenada Kryma' cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 µmol m-2 s-1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 µM BAP with 0.09 µM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in 'Crystal Fountain' (100%), 'Crimson Star' (100%), 'Nevesta' (97%), and 'Ay-Nor' (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

The complete chloroplast genome sequence of cultivated Prunus persica cv. 'Sovetskiy'.

The peach (Prunus persica L. Batsch) is one of the important stone fruit crops in the Crimea Peninsula and the southern part of Russia. The complete chloroplast genome of the peach cultivar 'Sovetskiy' is published in this paper. The chloroplast genome size is 157,756 bp. It contains 126 genes, including 81 protein-coding genes (PCGs), eight ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes. The chloroplast genome also contains a large single-copy region of 85,960 bp, a small single-copy (SSC) region of 19,045 bp, and two inverted repeats regions of 26,375 bp and 26,372 bp. The overall base composition of the genome in descending order is 31.2% - A, 32.1% - T, 18.7% - C, and 18.0% - G. The total GC content of the chloroplast genome is 36.7%. Maximum-likelihood phylogenetic analysis involving nine chloroplast genomes of the Prunus genus revealed a separate cluster for P. persica and its possible landrace - P. ferganensis.

Genetic Diversity of Peach Cultivars from the Collection of the Nikita Botanical Garden Based on SSR Markers.

The Nikita Botanical Garden (NBG) has a unique Prunus L. collection (peach, apricot, plum, cherry) comprising more than 3000 accessions. NBG is also a breeding center for stone fruits, including peach (Prunus persica (L.) Batsch). In the present study a set of 85 peach cultivars bred in NBG, Europe, and North America was analyzed using 12 SSR markers to assess their genetic diversity and relatedness. The detected polymorphism level was comparable to the previous estimates of genetic variability in peach cultivars. The average number of alleles per locus was 5.67, PIC value averaged 0.49, expected, and observed heterozygosity averaged 0.52 and 0.31, respectively. Among the detected alleles, 19 (27.94%) were rare and 12 (17.65%) were unique. All studied accessions except two could be identified with the used marker set. Cluster analysis revealed some groups according to the cultivars' pedigrees. No clear differentiation of the studied sample according to geographic origin or fruit characteristics of peach cultivars was revealed. The results provide valuable information for identification and rational management of the material preserved in the NBG peach collection.

The effect of neoadjuvant chemotherapy on the correlation of tumor-associated macrophages with CD31 and LYVE-1.

Angiogenesis and lymphangiogenesis play a crucial role in tumor growth, invasion and metastasis. Tumor-associated macrophages (TAM) induce both angiogenesis and lymphangiogenesis in mouse breast cancer models and positively correlate with these processes in human breast cancer patients. Neoadjuvant chemotherapy (NAC) is a widely used therapeutic option for cancer treatment. However, the effect of NAC on the distribution of TAM within intratumoral compartments and their correlation with angiogenesis and lymphangiogenesis remained unknown. In the present study we analyzed the effect of NAC on the distribution of CD68+ and stabilin-1+ TAM in five functionally distinct areas of human breast cancer and their correlations with microvessel density (MVD) and lymphatic microvessel density (LMVD), identified by CD31 and LYVE1, respectively. We found that NAC enhances blood vessel density in soft fibrous stroma and in coarse fibrous stroma. Without NAC the amount of CD68+ TAM in gaps of ductal tumor structures positively correlate with CD31+ microvessel density in soft fibrous stroma. NAC had enhancing effect on the amount of CD68+ TAM but not stabilin-1+ TAM in soft fibrous stroma. However, no correlation between TAM and CD31+ microvessel density was identified after NAC. NAC did not enhance the lymphatic microvessel density. But after NAC stabilin-1 expressing subpopulation of TAM positively correlated with expression of LYVE-1. We hypothesized that CD68+ TAM can support tumor angiogenesis primarily before NAC, while stabilin-1+ TAM can contribute to the maintenance of lymphatic microvessel density after NAC.

Development of Novel Monoclonal Antibodies for Evaluation of Transmembrane Prostate Androgen-Induced Protein 1 (TMEPAI) Expression Patterns in Gastric Cancer.

Transmembrane prostate androgen-induced protein 1 (TMEPAI) is a single-span membrane protein, functionally involved in transforming growth factor beta signaling pathway. The particular protein presented in cells in three isoforms, which differs in the length of the soluble N-terminal extracellular domain, making it challenging for the immunochemical recognition. By using quantitative real-time polymerase chain reaction, we identified significant upregulation of PMEPA1 gene expression in malignant tissues of patients with gastric adenocarcinoma. The main part of commercially available anti-TMEPAI antibodies are having polyclonal nature or not suitable for immunocytochemical localization of target protein in tissue specimens. Hence, we decide to generate a set of novel rat monoclonal antibodies (mAb) directed against conservative C-terminal cytoplasmic epitope. Immunoblotting analysis showed that monoclonal antibodies, 2E1, 6C6, and 10A7 were able to recognize specifically target protein in transiently transfected HEK293T and CHO-K1 cells. Especially established mAb, named 10A7, showed the excellent binding ability to target protein in immunohistochemistry. By using developed antibodies, we observed pronounced expression of TMEPAI in normal gastric epithelial cells while tumor cells from gastric adenomas, and adenocarcinoma samples were mostly negative for target protein expression. Also, we found that gastric epithelium cells lose the TMEPAI expression concurrently with severe dysplasia progression, which probably caused by a mechanism involving specific microRNA.

Role of the Immune Component of Tumor Microenvironment in the Efficiency of Cancer Treatment: Perspectives for the Personalized Therapy.

Despite significant progress in cancer diagnostics and development of novel therapeutic regimens, successful treatment of advanced forms of cancer is still a challenge and may require personalized therapeutic approaches. In this review, we analyzed major mechanisms responsible for tumor cells chemoresistance and emphasized that intratumor heterogeneity is a critical factor that limits efficiency of cancer treatment. Intratumor heterogeneity is caused by genomic instability in cancer cells, resulting in the selection of resistant clones. Moreover, cancer cells in solid tumors are surrounded by cellular and molecular microenvironment that actively influences tumor cell behavior. Local tumor microenvironment (TME) consisting of immune cells with diverse phenotypes and functions strongly contributes to intratumor heterogeneity and modulates responses to treatment. Thus, targeting specific components of TME is a novel treatment strategy that can improve the outcome of conventional anti-cancer therapy. Here, we discuss modern immunotherapeutic approaches based on targeting tumorinfiltrating immune cells including neutrophils, dendritic cells, NK cells, T cells, B cells and macrophages. Among those, tumor-associated macrophages (TAM) that display a pronounced heterogeneity and phenotypic plasticity appear to be a major component in the TME of solid tumors, and emerge as perspective targets for cancer immunotherapy. TAM intratumor heterogeneity and the possible existence of patient-specific phenotype signature generate the basis for the development of individualized TAM-based therapeutic approaches.

Tumor-associated macrophages in human breast cancer parenchyma negatively correlate with lymphatic metastasis after neoadjuvant chemotherapy.

Breast cancer is the leading cause of cancer death in women worldwide with high morbidity and mortality. Tumor-associated macrophages (TAM) are major innate immune cells in the tumor microenvironment controlling primary tumor growth and metastasis. Neoadjuvant chemotherapy (NACT) is a conventional pre-operative treatment for breast cancer. In the present study we examined the distribution of TAM in five distinct intratumoral morphological compartments of human breast cancer and their correlation with clinical parameters after NACT. Our data indicated that CD68+ but not stabilin-1+ TAM in areas with parenchymal elements negatively correlate with lymphatic metastasis after NACT. However, in cases where lymphatic metastases were detected (28 out of 50 analyzed samples) both amount of CD68+ and stabilin-1+ macrophages in the areas with coarse fibrous stroma directly correlated with the number of positive lymph nodes. In patients with complete response to the preoperative NACT the average score of CD68 expression in the areas with coarse fibrous stroma was lower compared with cases of a partial response and stable disease. We concluded that function of TAM after NACT depends on their intratumoral localization and local tumor microenvironment which plays an important role in polarization of macrophages towards tumor-suppressive or tumor-supportive types.

CD68+, but not stabilin-1+ tumor associated macrophages in gaps of ductal tumor structures negatively correlate with the lymphatic metastasis in human breast cancer.

Tumor associated macrophages (TAM) support tumor growth and metastasis in several animal models of breast cancer, and TAM amount is predictive for efficient tumor growth and metastatic spread via blood circulation. However, limited information is available about intratumoral TAM heterogeneity and functional role of TAM subpopulations in tumor progression. The aim of our study was to examine correlation of TAM presence in various morphological segments of human breast cancer with clinical parameters. Thirty six female patients with nonspecific invasive breast cancer T1-4N0-3M0 were included in the study. Morphological examination was performed using Carl Zeiss Axio Lab.A1 and MiraxMidiZeiss. Immunohistochemical and immunofluorescence/confocal microcopy analysis was used to detect CD68 and stabilin-1 in 5 different tumor segments: (1) areas with soft fibrous stroma; (2) areas with coarse fibrous stroma; (3) areas of maximum stromal-and-parenchymal relationship; (4) parenchymal elements; (5) gaps of ductal tumor structures. The highest expression of CD68 was in areas with soft fibrous stroma or areas of maximum stromal-and-parenchymal relationship (79%). The lowest expression of CD68 was in areas with coarse fiber stroma (23%). Inverse correlation of tumor size and expression of CD68 in gaps of tubular tumor structures was found (R=-0.67; p=0.02). In case of the lymph node metastases the average score of CD68 expression in ductal gaps tumor structures was lower (1.4±0.5) compared to negative lymph nodes case (3.1±1.0; F=10.9; p=0.007). Confocal microscopy identified 3 phenotypes of TAM: CD68+/stabilin-1-; CD68+/stabilin-1+ (over 50%); and CD68-/stabilin-1+. However, expression of stabilin-1 did not correlate with lymph node metastasis. We concluded, that increased amount of CD68+TAM in gaps of ductal tumor structures is protective against metastatic spread in regional lymph nodes.