Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Differential microRNA Expression Analysis in Patients with HPV-Infected Ovarian Neoplasms.

This study aimed to identify microRNAs (miRNAs) whose expression levels are altered by high-risk human papillomavirus (HR-HPV) infection in women with epithelial ovarian neoplasms. MiRNA expression was quantified by real-time polymerase chain reaction, while HR-HPV DNA was quantified using digital-droplet PCR. Analysis of 11 miRNAs demonstrated significantly lower hsa-miR-25-5p expression in HPV-infected compared to uninfected ovarian tissues (p = 0.0405), while differences in miRNA expression in corresponding serum were statistically insignificant. The expression of hsa-miR-218-5p in ovarian tumors was significantly higher in high-grade serous ovarian carcinoma (HGSOC) cases than in other neoplasms (p = 0.0166). In addition, hsa-miR-218-5p was significantly upregulated, whereas hsa-miR-191-5p was significantly downregulated in tissues with stage III/IV FIGO (p = 0.0009 and p = 0.0305, respectively). Using unsupervised clustering, we identified three unique patient groups with significantly varied frequencies of HPV16/18-positive samples and varied miRNA expression profiles. In multivariate analysis, high expression of hsa-miR-16-5p was an independent prognostic factor for poor overall survival (p = 0.0068). This preliminary analysis showed the changes in miRNA expression in ovarian neoplasms during HPV infection and those collected from HGSOCs or patients with advanced disease. This prospective study can provide new insights into the pathogenesis of ovarian neoplasms and host-virus interactions.

Molecular Identification of Strains within the Mycobacterium abscessus Complex and Determination of Resistance to Macrolides and Aminoglycosides.

One of the most relevant and pathogenic groups among the rapidly growing mycobacteria (RGM) is Mycobacterium abscessus complex (MABC) that includes three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. The aim of this study was the analysis of prevalence of MABC among other non-tuberculous mycobacteria isolated from patients in the Malopolska Region of Poland, between 2018 and 2021, as well as determination of their subspecies and molecular mechanisms of resistance to macrolides and aminoglycosides. The incidence of MABC was 5,4% (12/223). Eight strains were classified as M. abscessus subsp. abscessus, three as M. abscessus subsp. massiliense and one M. abscessus subsp. bolletii. Molecular analysis showed resistance to macrolides for eight strains of M. abscessus subsp. abscessus associated with erm(41)T28 gene mutations. One strain of M. abscessus subsp. abscessus showed resistance to macrolides (two mutations simultaneously: in erm(41)T28 and rrl genes) and aminoglycosides (point mutation in rrs gene). One strain of M. abscessus subs. bolletii was resistant to macrolides (erm(41)T28 mutation), whereas presented no mutations for aminoglycosides. M. abscessus subsp. massiliense reveal no mutations. High clarithromycin resistance of M. abscessus, determines the urgent need for susceptibility-based treatment. Molecular determination of resistance mechanisms to aminoglycosides and macrolides enables fast and accurate targeted treatment implementation.

New SDS-Based Polyelectrolyte Multicore Nanocarriers for Paclitaxel Delivery-Synthesis, Characterization, and Activity against Breast Cancer Cells.

The low distribution of hydrophobic anticancer drugs in patients is one of the biggest limitations during conventional chemotherapy. SDS-based polyelectrolyte multicore nanocarriers (NCs) prepared according to the layer by layer (LbL) procedure can release paclitaxel (PTX), and selectively kill cancer cells. Our main objective was to verify the antitumor properties of PTX-loaded NCs and to examine whether the drug encapsulated in these NCs retained its cytotoxic properties. The cytotoxicity of the prepared nanosystems was tested on MCF-7 and MDA-MB-231 tumour cells and the non-cancerous HMEC-1 cell line in vitro. Confocal microscopy, spectrophotometry, spectrofluorimetry, flow cytometry, and RT PCR techniques were used to define the typical hallmarks of apoptosis. It was demonstrated that PTX encapsulated in the tested NCs exhibited similar cytotoxicity to the free drug, especially in the triple negative breast cancer model. Moreover, SDS/PLL/PTX and SDS/PLL/PGA/PTX significantly reduced DNA synthesis. In addition, PTX-loaded NCs triggered apoptosis and upregulated the transcription of Bax, AIF, cytochrome-c, and caspase-3 mRNA. Our data demonstrate that these novel polyelectrolyte multicore NCs coated with PLL or PLL/PGA are good candidates for delivering PTX. Our discoveries have prominent implications for the possible choice of newly synthesized, SDS-based polyelectrolyte multicore NCs in different anticancer therapeutic applications.

Prevalence of Closely Related Candida albicans Species among Patients with Vulvovaginal Candidiasis in Southern Poland Based on the hwp1 Gene Amplification.

Candida albicans remains the most common species isolated from women with vulvovaginal candidiasis. However, closely related species such as Candida africana and Candida dubliniensis may also occur, although they are often misidentified. The aim of the study was to confirm the phenotypic identification of C. albicans and its closely related species isolated from women with genital tract infections by amplification of the hwp1 (hyphal wall protein 1) gene in a PCR assay. We report a detailed molecular identification of C. albicans and its closely related species among 326 patients in the Malopolska region, Poland. Initial phenotypic identifications were confirmed by amplification of the hwp1 gene. Based on molecular analysis, we revealed 307 strains (94.17%) as C. albicans and 17 as C. dubliniensis (5.22%). No strain of C. africana was detected. Two patients h ad co-infection with C. albicans and C. dubliniensis (0.61%). A PCR assay targeting the hwp1 gene was reliable for correctly identifying species among the C. albicans complex.

Clonal transcriptomics identifies mechanisms of chemoresistance and empowers rational design of combination therapies.

Tumour heterogeneity is thought to be a major barrier to successful cancer treatment due to the presence of drug resistant clonal lineages. However, identifying the characteristics of such lineages that underpin resistance to therapy has remained challenging. Here, we utilise clonal transcriptomics with WILD-seq; Wholistic Interrogation of Lineage Dynamics by sequencing, in mouse models of triple-negative breast cancer (TNBC) to understand response and resistance to therapy, including BET bromodomain inhibition and taxane-based chemotherapy. These analyses revealed oxidative stress protection by NRF2 as a major mechanism of taxane resistance and led to the discovery that our tumour models are collaterally sensitive to asparagine deprivation therapy using the clinical stage drug L-asparaginase after frontline treatment with docetaxel. In summary, clonal transcriptomics with WILD-seq identifies mechanisms of resistance to chemotherapy that are also operative in patients and pin points asparagine bioavailability as a druggable vulnerability of taxane-resistant lineages.

Cancer begins when a cell multiplies again and again to form a tumour. By the time that tumour measures a centimetre across, it can contain upwards of a hundred million cells. And even though they all came from the same ancestor, they are far from identical. The tumour's family tree has many branches, and each one responds differently to treatment. If some are susceptible to a drug the cells die, the tumour shrinks, and the therapy will appear to be successful. But, if even a small number of cancer cells survive, they will regrow, often more persistently, causing a relapse. Identifying resistant cells, their characteristics, and how to kill them has been challenging due to a lack of good animal models. One way to keep track of a cancer family tree is to insert so-called genetic barcodes into the ancestral cells. As the tumour grows, the cells will pass the barcodes to their descendants. Scientists do this by using viruses that naturally paste their genes into the cells they infect. Applying this technique to an animal model of cancer could reveal which genes allow some cells to survive, and how to overcome them. Wild, Cannell et al. developed a genetic barcoding system called WILD-seq and used it to track all the cells in a mouse tumour. The mice received the same drugs used to treat patients with breast cancer. By scanning the genetic barcodes using recently developed single cell sequencing technologies, Wild, Cannell et al. were able to identify and count each type of cancer cell and work out which genes they were using. This revealed which cells the standard treatment could not kill and exposed their genetic weaknesses. Wild, Cannell et al. used this information to target the cells with a drug currently used to treat leukaemia. The drug identified by this new genetic barcoding approach is already licensed for use in humans. Further investigation could reveal whether it might help to shrink breast tumours that do not respond to standard therapy. Similar experiments could uncover more information about how other types of tumour evolve too.

The Toll-like Receptor 4 Polymorphism Asp299Gly Is Associated with an Increased Risk of Ovarian Cancer.

Ovarian cancer (OC) is one of the most common cancers threatening women's lives around the world. Epithelial ovarian tumors represent the most common ovarian neoplasms. Most OC patients are diagnosed at the advanced stage, and there is an urgent need to identify novel biomarkers of the disease. Single-nucleotide polymorphisms (SNPs) in TLR genes may serve as crucial markers of cancer susceptibility. We investigated the frequency of TLR polymorphisms in a group of 200 women, including 70 with OC. Four SNPs, two each in TLR4 (rs4986790 and rs4986791) and TLR9 (rs187084 and rs5743836), were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The digested fragments were separated and identified by multicapillary electrophoresis. The load quantification of human papillomavirus (HPV) types 16/18 was determined using a digital droplet PCR method. We found an increased frequency of heterozygous genotype and minor allele of the TLR4 rs4986790 SNP in women with OC compared with healthy controls, and this result remained highly significant after Bonferroni's correction for multiple testing (p < 0.0001). No evidence of linkage disequilibrium was found with any of the examined TLR SNPs. The findings suggest that the TLR4 Asp299Gly polymorphism could be a genetic risk factor for the development of OC.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells.

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products toward the clinic.

Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation.

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

Resolving the immune landscape of human prostate at a single-cell level in health and cancer.

The prostate gland produces prostatic fluid, high in zinc and citrate and essential for the maintenance of spermatozoa. Prostate cancer is a common condition with limited treatment efficacy in castration-resistant metastatic disease, including with immune checkpoint inhibitors. Using single-cell RNA-sequencing to perform an unbiased assessment of the cellular landscape of human prostate, we identify a subset of tumor-enriched androgen receptor-negative luminal epithelial cells with increased expression of cancer-associated genes. We also find a variety of innate and adaptive immune cells in normal prostate that were transcriptionally perturbed in prostate cancer. An exception is a prostate-specific, zinc transporter-expressing macrophage population (MAC-MT) that contributes to tissue zinc accumulation in homeostasis but shows enhanced inflammatory gene expression in tumors, including T cell-recruiting chemokines. Remarkably, enrichment of the MAC-MT signature in cancer biopsies is associated with improved disease-free survival, suggesting beneficial antitumor functions.

Interaction of Adenosine, Modified Using Carborane Clusters, with Ovarian Cancer Cells: A New Anticancer Approach against Chemoresistance.

Platinum compounds remain the first-line drugs for the treatment of most lethal gynecological malignancies and ovarian cancers. Acquired platinum resistance remains a major challenge in gynecological oncology. Considering the unique physicochemical properties of the metallacarboranes modifier and the significant role of nucleoside derivatives as anticancer antimetabolites, we designed and synthesized a set of adenosine conjugates with metallacarboranes containing iron, cobalt, or chromium as semi-abiotic compounds that influence the cisplatin sensitivity of ovarian cancer cells. Adherent cultures of ovarian carcinoma cell lines and multicellular spheroids, ranging from sensitive to highly resistant including experimental cell lines "not responding" to platinum drugs were used. Iron-containing metallacarborane conjugates showed the best anticancer activity, especially against resistant cells. Compound modified at the C2' nucleoside position showed the best activity in resistant cancer cells and highly resistant cancer spheroids exposed to cisplatin, increasing cell cycle arrest, apoptosis or necrosis, and reactive oxygen species production. Moreover, it showed high cellular accumulation and did not induce cross-resistance to cisplatin, carboplatin, doxorubicin, paclitaxel, or gemcitabine in long-term cultures. The reference nido-carborane derivative (no metal ions) and unmodified nucleosides were not as effective. These findings indicate that metallacarborane modification of adenosine may sensitize ovarian cancer cells to cisplatin in combination treatment.

Single-cell multi-omics analysis of the immune response in COVID-19.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors.

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

The Medial Femoral Condyle Flap in the Pediatric Patient.

BACKGROUND: The use of free flaps from the medial femoral condyle has grown in popularity and is now a workhorse in the reconstruction of skeletal defects. The utility of this technique has not yet been described for the pediatric patient population. The authors present their series of pediatric patients who underwent surgery using a medial femoral condyle free flap or a variant thereof in skeletal reconstruction and demonstrate the efficacy of this technique in this population. METHODS: A multi-institutional retrospective review of patients aged 18 years or younger who required a medial femoral condyle flap for skeletal reconstruction was undertaken. Operative technique, radiographs, and clinical outcomes were recorded. A novel technique (Innocenti) was used to avoid the distal femoral physis in which a Kirschner wire was placed under fluoroscopic guidance just proximal to the growth plate. RESULTS: Thirteen patients met inclusion criteria, with an average age of 14.7 years (range, 7 to 18 years) and mean follow-up of 28 months (range, 3 to 120 months). Six were skeletally immature at the time of medial femoral condyle harvest, with the last patient having organic bone disease, putting her at risk for pathologic fracture. All 13 patients achieved bony union, and no patients suffered pathologic fractures or physeal injuries; no patients developed length discrepancies. CONCLUSIONS: The authors present the first series of corticocancellous medial femoral condyle free flaps in the pediatric population along with a novel technique to avoid injury to the physis in skeletally immature patients. This technique is effective for a variety of skeletal defects or nonunions and is safe for growing patients without causing physeal arrest or growth disturbance. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Post-mastectomy sensory recovery and restoration.

Breast sensation has recently become an integral aspect of the reconstructive goal after mastectomy and is an important consideration for many patients. Neurotization techniques using primary coaptation, autograft, allograft, or nerve conduit have been used for autologous flaps, such as the deep inferior epigastric perforator (DIEP) flap. Outcomes have shown improved sensation and faster sensory recovery in the flap skin in immediate neurotized DIEP flap breast reconstructions compared to delayed reconstruction. Breast flap neurotization during reconstruction is a rapid and simple procedure with minimal morbidity. An improved understanding of breast anatomy and innovative modifications to breast reconstruction have made the restoration of breast sensation achievable, and promising results have been obtained with respect to sensory return and patient satisfaction.

CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood-Brain Barrier Models.

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood-brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood-brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells.

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin-transferrin (DOX-Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX-Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX-Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX-Tf conjugate.

Quantifying Free Fibula Flap Growth After Pediatric Mandibular Reconstruction.

INTRODUCTION: The free fibula flap (FFF) is a preferred option for adult mandibular reconstruction. Due to skeletal immaturity, its routine use in pediatric patients remains in question. Inconsistencies regarding the ability of the FFF to grow in concordance with the patients' natural growth currently exist in the literature. The purpose of this report is to quantify mandibular growth in a young patient undergoing partial hemi-mandibular reconstruction with a FFF utilizing advanced three-dimensional software. METHODS: A 2-year old underwent left hemi-mandibular reconstruction with a FFF following resection of a desmoid tumor. The condyle was preserved. Using 3D software, changes in mandibular growth and morphology were evaluated based on preoperative (2.1 years old) and postoperative (2.5 years and 5.2 years old) computed tomography imaging. RESULTS: Mandibular growth occurred throughout the mandible in both postoperative evaluations. Greatest growth was seen in the ramus height. Fibula growth was also seen when comparing measurements to the virtual surgical planning guide. A novel parts comparison analysis revealed the greatest growth potential occurred at the condyle. CONCLUSION: Providing an objective evaluation using 3D software, we have demonstrated growth throughout the reconstructed mandible, with greatest growth occurring at the preserved condyle. Despite scientific limitations of our study, the potential for mandibular growth appears to remain after FFF reconstruction, offering successful functional and cosmetic outcomes.