The biomarkers mentioned above are widely validated but are found within the cell, and therefore require cell isolation (and usually destruction) for analysis. For clinical implementation, it is more useful to rely on molecules that are secreted in the adjacent extracellular area and can be detected without disturb- ing the cells in human subjects. The various SASP components, such as inflammatory molecules (cytokines and chemokines, ex- tracellular damage-associated alarmin high-mobility group box 1 [HMGB1] protein), growth factors and regulators (transform- ing growth factor beta [TGF𝛽 ], insulin-like growth factor-binding

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formin (TAME) Biomarkers Workgroup established a framework for the selection of blood-based biomarkers for next-generation geroscience clinical trials.[65]

Data on the quantity of active stem cells in other organs and how their abundance and functionality change with age would be very pertinent, especially in the context of novel therapeutic approaches, such as direct reprogramming of differentiated tis- sue cells[58] or rejuvenation of dysfunctional adult stem cells,[59] designed to maintain robust stem cell pools.

3.2. Inflammatory Markers

Aging is often associated with the dysregulation of the immune system, leading to chronic systemic low-grade inflammation. Cir- culating proinflammatory mediators, such as cytokines and their 𝛽 𝛼 soluble receptors (IL-6, IL-8, IL-1 , TNF- , TNFRs), C-X-C motif chemokines (CXCL9, CXCL10), C-C motif chemokines (CCL2, С also known as MCP-1, CL11 also known as eotaxin), CX3C mo- tif chemokine (CX3CL1) as well as acute phase C-reactive protein produced inliverinresponse toinflammationare amongpromis- ing biomarkers of aging.[1] Due to that fact that many of these mediators are classical SASP components, cellular senescence is likely to participate in inflammaging, albeit the true extent to which senescence contributes to systemic markers as compared to other inflammatory drivers remains unknown. Additional in- formation regarding age-related changes in immune function can be obtained from immune-phenotype profiling (lymphocyte subset panel, CD4:CD8 T cell ratio).[6]

2.6. Altered Intercellular Communication

Although cell aging is assumed to be the main cause of over- all human aging, our body consists not only of cells, but also of the extracellular space, which is likewise affected by time. Cross- linking of extracellular matrix molecules, especially collagen, and accumulation of the waste products, such as AGEs, make the cell environment rigid, impeding cell–cell interaction and cell migra- tion within tissues.[60] In contrast to the intracellular space, which has efficient intracellular mechanisms of waste recycling, the ex- tracellular space lacks such machinery and disposal of garbage outside the cell is a serious age-related problem. Regulatory and effector molecules which are secreted into an immediate environ- ment, such as various SASP components, or found in circulating body fluids, such as hormones, systemic inflammatory markers, and circulating miRNAs, also contribute to altered cell commu- nication and activity with age.

Despite no standard cytokine signature exists in age-related chronic inflammation in contrast to the acute inflammatory re- sponse, for which a number of secreted molecules have been val- idated, a deep-learning based inflammatory aging (iAGE) clock has been recently created (Table S1, Supporting Information).[66] The clock is based on circulating immune protein data of 50 cy- tokines, chemokines, and growth factors, with the ability to pre- dict multimorbidity with 0.41 mean absolute error. In centenari- ans, iAge was on average, 40 years lower than their corresponding chronological age, witch chemokine CXCL9 and down-regulated Sirtuin-3 as the strongest contributors.

3. Systemic Biomarkers

Going further up in the hierarchical structure of the human body, the aging process can be ultimately observed at the sys- tem level as well. The functional decline in specific tissues and organs due to the accumulation of aged cells, complicated by in- trasystem/intersystem interactions and by environmental factors (e.g., diet, lifestyle) may be best measured by systemic molecular biomarkers. Although these biomarkers reflect deleterious con- sequences of aging, rather than primary causes such as molecu- lar damage, they provide the essential input to developing the pre- diction models for biological age comprising information from multiple systems throughout the body.[61]

3.3. Metabolic Markers

Altered energy metabolism is another major feature of aging. At the cellular level, it occurs predominantly due to the disruption in nutrients-sensing pathways highly involved in aging progres- sion, such as the mechanistic target of rapamycin (mTOR) (nu- trient sensor), the adenosine monophosphate (AMP)-activated protein kinase (AMP sensor), the protein deacetylase sirtuins + (regulated by NAD ), and insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathways.[67] Consequent impairment in cellular utilization of metabolic substrates, especially glucose, leads to decreased energy production. The level of blood glucose is primarily determined by insulin secretion, which, in turn, is affected by gradual decline of 𝛽 -cell function and mass.[68] Glu- cose uptake and oxidation are often reduced later in life with the onset and progression of peripheral insulin resistance, partic- ularly in skeletal muscle and adipose tissue, which can lead to weight loss and frailty observed in the elderly[34]; population- based studies revealed that centenarians have higher insulin sensitivity and preserved 𝛽 -cell function than younger controls[69] suggesting the importance of normal energy metabolism for healthy aging. Biomarkers of dysregulated glucose metabolism, especially glycated haemoglobin HbA1C, despite being typically

3.1. Classical Blood-Based Biomarkers